CA2089752A1 - Production of human lysozyme in methylotrophic yeast cells and efficient secretion therefrom - Google Patents

Abstract

Human lysozyme (HLZ), a naturally occurring, relatively short, single chain polypeptide, is prepared by growing methylotrophic yeast transformants containing in their genome at least one copy of a first expression cassette comprising a DNA sequence operably encoding HLZ, in operational association with a DNA sequence encoding the S. cerevisiae alpha mating factor pre-pro secretion signal sequence (including the proteolytic processing site: lys-arg), and optionally further including a second expression cassette, comprising a DNA sequence operably encoding HLZ, in operational association with a DNA sequence encoding the native human lysozyme, secretion signal sequence, wherein both the coding sequence(s) and the secretion signal sequence(s) are maintained under the regulation of promoter region(s) of gene(s) of methylotrophic yeast, under conditions allowing expression of said DNA sequences, and secretion of HLZ into the culture medium. Also disclosed are novel DNA fragments and novel recombinant yeast strains which are useful in the practise of the present invention.

Description

92/~4~1 PC1/1591/063 PRODUCTION OF HUMAN LYSO~YME IN METHYLOTROPHIC
YEAST CELLS AND EFFICIENT SECRETION THEREFROM

Field of the Invention This invention relates to a process of ' recombinant DNA technology for producing human lysozyme S (HLZ) peptides in methylotrophic yeast such as Pichia pastoris. The invention further relates to the methylotrophic yeast transformants, DNA fragments and expression vectors used for their production and cultures containing same.

Back~round of the Invention '; ' Lysozymes are basic enzymes which exhibit anti-bacterial action directly, as a result of their ability to lyse bacterial cells, and indirectly, as A
result of their ability to produce a stimulatory effect upon the phagocytic activity of polymorphonuclear leukocytes and macrophages (Jolles et al., Mol. and Cell. Biochem. 63: 165 (1984)). Lysozymes exist in many tissues and secretions of humans, other vertebrates and invertebrates, as well as in plants, ; bacteria and phage. In performing their primary function of protecting organisms against bacterial infection, lysozymes, which are also known as 1,4-~-N-acetylmuramidases, cleave the glycosidic bond between the C-l of N-acetylmuramic acid and the C-4 of N-acetylgulcosamine in the bacterial peptidoglycan, a polysaccharide of amino sugars attached to short cross-linked peptides which is a component of bacterial cell walls. Gram-negative bacteria have cell walls which contain mono- or bi-layered peptidoglycan whereas gram-; positive bacteria possess cell walls which contain :`

WO92/04~1 PCT/~'S91/06326 5~ .

highly complex multi-layered peptidoglycan. As a result of the above-described cleavage, all such bacteria lyse and, consequently, die. Some lysozymes also display a more or less pronounced chitinase activity, corresponding to a random hydrolysis of 1,4-~-N-acetyl-glucosamine linkages in chitin, and consequently have the additional capacity of protecting organisms against a large number of chitin-covered : pathogens. A slight esterase activity of lysozymes has lo also been reported.
Due to their bacteriolytic activity, lysozymes are employed, by themselves and in combination with other components (e.g., lactotransferrin (which inhibits the growth of certain microorganisms by chelating iron), complement, antibodies, vitamins, other enzymes and various antibiotics (e.g., tetracycline and bacitracin), as antimicrobial agents, as preservatives for foods (e.g., cheese, sausage and marine products), as ripening agents for cheese, and in various other applications.
Since lysozymes also possess the ability to indirectly stimulate the production of antibodies against a variety of antigens, such enzymes may also be employed to enhance resistance against infection.
Lysozymes of the c, or "chicken", type contain 129-130 amino acids in their mature, secreted forms. Forty of these 129-130 amino acids have been found to be invariant among different species. Two of the several carboxyl groups of lysozyme molecules ~corresponding to the Glu-35 and Asp-52 of the chicken egg white lysozyme amino acid sequence) which participate in the enzyme's catalytic activity, and which are essential for lysozyme activity, occur in similar positions in all c-type lysozymes. A third . , , , . .. . .. . . . . .
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~92/04441 PCT/US91/06326 carboxyl group (corresponding to Asp-101 in chicken egg white lysozyme), which is involved in a substrate binding interaction, occurs in most c-type lysozymes.
The eight half-cysteine residues of all of the c-type lysozymes are invariant. The disulfide bonds formed by the cysteines play an important role in the formation and maintenance of the enzymes' secondary and tertiary structures. The three-dimensional structures, and processes of folding to form these structures upon translation of mRNAs, are thought to be closely similar for all lysozyme c's. The complete primary structures are known for the mature lysozyme c's obtained from the following sources: (1) hen, quail, turkey, guinea fowl, duck, pheasant, chachalaca and chicken egg whites; (2) human milk and urine; (3) moth; (4) baboon, rat, and bovine stomach; and (5) T2 and T4 phage. DNA
sequences encoding mature human milk lysozyme c are also known. See European Patent Application Publication Nos. 0 181 634, 0 208 472, and 0 222 366.
Considering the many uses of lysozymes, a ready supply of human lysozyme (HLZ), such as that which would result from fermentation of HLZ-expressing recombinant organisms, will be of great value to the medical and biotechnology fields.
Since isolation from natural sources is technically difficult, expensive, and time consuming, recent efforts have centered on the development of efficient recombinant methods for the production of HLZ.
Of the hosts widely used for the recombinant production of heterologous proteins, probably E. coli and Saccharomyces cerevisiae (Baker's yeast) are the best understood. However, E. coli tends to produce disulfide-bonded proteins such as HLZ in their reduced .

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W092/04~l PCT/US9t/06326 _4_ ; forms which frequently are not stable in the presence of endogenous bacterial proteases, and which tend to aggregate into inactive complexes. See, for example, Muraki et al., in Aaric. Biol. Chem., 49: 2829-2831 (1985). Attempts to overcome this problem, e.g., by employing a suitable leader sequence in order to produce soluble HLZ which could be readily recovered from the cell broth, will frequently result in other inconveniences, especially during purification of the product, since the bulk of the desired protein may become associated with the cell paste.
, The production of heterologous proteins by - secretion to the culture medium mediated by signal sequences has been described for many organisms, , 15 including various Aspergillus species, Saccharomyces cerevisiae, and various types of mammalian cells. In these species, both native (i.e., intra-generic) and mammalian signal sequences have been demonstrated to be , capable of directing secretion of certain heterologous ; 20 proteins into the growth media. However, each of these host systQm6 has various disadvantages.
For example, Aspergillus strains secrete large quantities of endogenous proteins into the growth media, thus significantly increasing the complexity and 25 the expense of purifying a desired heterologous protein ~` product. The productivity of heterologous protein production by secretion from S. cerevisiae appears to be somewhat limited, for those proteins which can be secreted at all. A major disadvantage associated with ;, 30 mammalian cell hosts is the difficulty of, and large , expense associated with, maintaining such host systems ~3 and culturing such hosts on a large scale.
Yeasts can offer clear advantages over bacteria in the production of heterologous proteins, .; .

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~ W092/04Wl PCT/~;S91/06326 ,', Ç~ 20 which include their ability to secrete heterologous proteins into the culture medium. Secretion of proteins from cells is generally superior to production of proteins in the cytoplasm. Secreted products are obtained in a higher degree of initial purity; and further purification of the secreted products is made easier by the absence of cellular debris. In the case of sulfhydryl-rich proteins, there is another compelling reason for the development of eukaryotic hosts capable of secreting such proteins into the culture medium: their correct tertiary structure is produced and maintained via disulfide bonds. This is because the secretory pathway of the cell and the extracellular medium are oxidizing environments which can support disulfide bond formation [Smith, et al., Science, 229, 1219 (1985)]; whereas, in contrast, the cytoplasm is a reducing environment in which disulfide bonds cannot form. Upon cell brea~age, too rapid formation of disulfide linkages can result in random disulfide bond formation. Consequently, production of sulfhydryl-rich proteins, such as HLZ, containing appropriately formed disulfide bonds, can perhaps best be achieved by transit through the secretory pathway.
A number of publications have appeared relating to the recombinant production of human lysozyme in the yeast, S. cerevisiae. For example, European Patent Application 255,233 describes the ; production of human lysozyme by cultivating a cell ; transformed with a DNA sequence which codes for hen egg white lysozyme signal peptide bound to the 5' end of a DNA segment coding for human lysozyme. Cultivation of S. cerevisiae transformed with this construct produced only a few mg per liter of human lysozyme, with about .

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European Patent Application 251,730 similarly describes the production of human lysozyme employing a synthetic signal peptide which is said to be superior to that of hen egg white lysozyme for secretive production of human lysozyme. The production of only about 5 mg/~ of secreted HLZ is all that is demonstrated, however.
Other references which disclose the secreted production of HLZ by the yeast S. cerevisiae include Suzuki et al., 14th Int. Conf. on Yeast Genetics and Molecular Bioloav, p. E34 (1988: describes yeast mutants which oversecrete HLZ, relative to wild-type parent strains--employing DNA seaments coding for chicken lysozyme signal sequence and the mature portion of human lysozyme); Jigami et al., Gene 43: 273-279 ~1986; describes the use of a multicopy plasmid ~; containing a synthetic chicken lysozyme signal sequence ;;~ 20 and a synthetic HLZ gene to direct the synthesis and secretion of HLZ in S. cerevisiae); Nagahora et ~
FLBS Letters, ~ 329-332 (1988; describes the effect on secretion of human lysozyme by altering the cleavage site of the native chicken lysozy~e signal sequence associated therewith); Taniyama et al., Biochemical and Biophysical Research Communications, 152: 962-967 (1988; examines the role of disulfide bonds in folding and secretion of human lysozyme in S. cerevisiae);
;~ Yoshimura et al., Biochemical and BioDhvsical Research Communications, 145: 712-718 (1987; compares the differences between secreted HLZ produced by S.
cerevisiae and B. subtilis); Yamamoto et al., ~; ~iochemical and BioDhYsical Research Communications, 149: 431-436 (1987; describes the secretion of HLZ by . ' .
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,, ,; , ", ~: . .. -~ . . . . , ~ 92tO4441 PCI /l 'S91/06326 .:', ;l S. cerevisiae using engineered forms of the signal sequence of chicken lysozyme); Yoshimura et al., Biochemical and Biophysical Research Communications, . 150: 794-801 (1988; describes the isolation of cDNA
encoding human lysozyme (including the human lysozyme signal sequence) from a human placenta library.
Expression and secretion of several mg/L of HLZ from this cDNA in S. cerevisiae is also described); Castanon et al., Gene, 66: 223-234 (1988; discloses the isolation of cDNA clones encoding human lysozyme from a human histiocytic cell line and a human placenta cDNA
library. Expression and secretion (up to about 15 mg/L) of human lysozyme in S. cerevisiae was achieved by placing the cloned cDNA under the control of the S.
cerevisiae ADHl gene promoter and the alpha-factor peptide leader sequence); Hayakawa et al., Gene, 56:
53-59 (1987; report the production of an insoluble and biologically inactive form of recombinant human , lysozyme that required solubilization and oxidative 20 renaturation to produce biologically active material);
and European Patent Application No. 208,~72 (describes the non-secreted production of human lysozyme by S.
cerevisiae).
Other publications dealing with the 25 recombinant production of HLZ include Japanese Application No. 2027989 (1/30/90; priority JP 88 177754 (7/15/88); assigned to Sumitomo Chem. Ind. KK);
Japanese Application No. 2027988 (1/30/90; priority JP
88 177753 (7/15/88); assigned to Sumitomo Chem. Ind.
30 KK); Japanese Application No. 20 05879 (1/10/90;
priority JP 88 151106 (6/21/88); assigned ~o Takeda Chemical Ind. KK); Japanese Application No. 1074989 ", (3/20/89; priority JP 87 229752 (9/16/87); assigned to ~ Takeda Chemical Ind. KK); Japanese Application No. ~3 ,. ' ~

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wos2/o~ 9~ PCT/US91/063 052882 (3/7/88; priority JP 86 196226 (8/21/86), assigned to Sumitomo Chem. Ind. KK); Japanese ~ ~
Application No. 83 052881 (3/7/88; priority JP 86 ~-195885 (8/20/86); assigned to Sumitomo Chem. Ind. KK);
Japanese Application No. 83 052876 (3/7/88; priority JP
86 195886 (8/20/86); assigned to Sumitomo Chem. Ind.
KK); Japanese Application No. 62 166884 (7/23/87;
priority JP 86 5092 (1/16/86); assigned to Takeda Chemical Ind KK); EP 222,366 (5/20/87; assigned to Boehninger Ingelheim); and Japanese Application ~o. 61 078387 (4/21/86; priority JP 84 201367 (9/26/84);
assigned to Agency of Ind. Sci. Tech. and Sumitomo Chemical KK).
Some polypeptides which are expressed in, and secreted from, heterologous hosts, however, exhibit no, or reduced, levels of biological activity in comparison to their naturally occurring counterparts. This reduced biological activity may be the result of a number of factors, including the inability of the polypeptide to pass into and through the host system' 5 ;,-~ secretory apparatus without activity-reducing degradation or cleavage and to properly fold and form disulfide bonds during the secretion process.
In view of the above-described problems, as well as the problems frequently encountered with up scaling the production of heterologous proteins in autonomous plasmid-based yeast systems, such as S.
cerevisiae, and the low expression levels of human lysozyme achieved in reported recombinant wor~, no motivation is provided by the art for one to further pursue the production of HLZ employing presently available recombinant systems. To overcome the major ;~. problems associated with the expression of recombinant gene products in S. cerevisiae (e.g., loss of selection ,:`
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~ 92/0~1 PCT/~'S91/06326 ;. 2~897~2 ,', _g_ for plasmid maintenance and problems concerning plasmid distribution, copy number and stability in fermentors ; operated at high cell density), a yeast expression system based on methylotrophic yeast, such as for example, Pichia pastoris, has been developed.
Methylotrophic yeasts have been found to be particularly favorable for use as host systems for the large-scale production of those heterologous proteins which they are capable of secreting into the culture media at significant levels in biologically active form. Methylotrophic yeast, e.g., P. pastoris, are ,1 readily adaptable to continuous industrial-scale .... ~ .
; fermentat$on processing, whereby the yeasts grow to high cell densities in a defined and inexpensive fermentation medium. With P. pastoris, for example, production levels usually scale up from shake-flask ' cultures to large fermentor cultures. Simple culture media which are inexpensive and free of undefined i ingredients, which can be potential sources of pyrogens and toxins, can be used for such yeast. Moreover, ~ since many critical functions of yeasts, such as ,d oxidative phosphorylation, are performed within ,t3 organelles, such functions are not, as they are in prokaryotic hosts, directly exposed to the possible deleterious effects of production of polypeptides foreign to the host cells. Also, since yeasts are 1 eukaryotes, their intracellular environment tends to be ~' more suitable than that of prokaryotes for the correct folding of eukaryotic proteins. ln addition, the cultivation of yeasts, particularly P. pastoris, is easier than that of most other host systems.
" Contamination of P. pastoris cultures growing on ;~ methanol can more easily be prevented than that of ,~ cultures of other types of hosts, thereby increasing 'I ., .
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the reliability and safety of the heterologous polypeptide products.
A key feature of expression systems based on methylotrophic yeast lies with the promoter employed to drive heterologous gene~expression. This promoter, which is derived from a methanol-responsive gene of a methylotrophic yeast, is frequently highly expressed ; and tightly regulated (see, e.g., U.S. Patent No.
;~ 4,855,231). Another key feature of expression systems based on methylotrophic yeast is the ability of expression cassettes to stably integrate into the genome of the methylotrophic yeast host, thus significantly decreasing the chance of vector loss.
The methylotrophic yeast, P. pastoris, has been used for the successful production of selected heterologous proteins, e.g., hepatitis B surface antigen [Cregg et al., Bio/TechnoloaY 5, 479 (1987)], lysozyme [see WO 89/03907, published 5/18/89; assigned ~! to Salk Institute Biotechnology/Industrial Associates;
describes bovine lysozyme secretion promoted by the ~ native lysozyme signal sequence~ and invertase lDigan ', et al., Developments in Industrial Microbloloay 29, 59 ~1988); Tschopp et al., Bio/TechnoloaY 5, 1305 (1987)~.
Endeavors to produce other heterologous gene products in Pichia, however, especially by secretion, have given mixed results. At the present level of understanding of methylotrophic yeast expression systems, it is unpredictable whether a given gene can be expressed to an appreciable level in such yeast or whether the yeast ` 30 host will tolerate the presence of the recombinant gene product in its cells. Further, it is especially ' difficult to foresee if a particular protein will be ' secreted by the methylotrophic yeast host, and if it is, at what efficiency. Even for the non-,, .

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methylotrophic yeast, S. cerevisiae, which has been considerably more extensively studied than P. pastoris, the mechanism of protein secretion is not well defined and understood.

; Summary of the Invention In accordance with the present invention, we have developed an improved expression system for the production of biologically active human lysozyme (HLZ) molecules. The present invention provides a powerful method for the production of secreted HLZ peptides in methylotrophic yeast. In addition, the invention method can easily be scaled up from shake-flask cultures to large scale fermentors with no loss in HLZ
productivity. Moreover, the invention method can readily be scaled up without the need for making major changes in the fermentation conditions used for the large scale growth of the transformed strains relative to the conditions used for small scale growth of transformed strains.
We have surpri6ingly found that HLZ peptides can very efficiently be produced in, and secreted from, methylotrophic yeast, such as, for example, P.
pastoris. This is accomplished by transforming a methylotrophic yeast with, and preferably integrating into the yeast genome, at least one copy of a first DNA
sequence operably encoding an HLZ peptide, wherein said first DNA sequence is operably associated with a second DNA sequence encoding the S. cerevisiae alpha-mating factor (AMF) pre-pro sequence (including the proteolytic processing site: lys-arg)~ and wherein both of said DNA sequences are under the regulation of a methanol responsive promoter region of a gene of a .

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methylotrophic yeast. Methylotrophic yeast cells containing in their genome at least one copy of these DNA sequences efficiently produce and secrete biologically active HLZ peptides into the medium.
The present invention is directed to the above aspects and all associated methods and means for accomplishing such. For example, the invention includes the technology requisite to suitable growth of the methylotrophic yeast host cells, fermentation, and 10 isolation and purification of the HLZ gene product.
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Brief Descript~on of the Drawinas Figure 1 provides the nucleotide sequence of 15 the human lysozyme gene employed in the examples.
Figure 2 is a restriction map of plasmid pAO815.
Figure 3 is a restriction map of plasmid pHLZ103.
Figure 4 is a restriction map of plasmid pHLZ105.
Figure 5 is a restriction map of plasmid pAHZl06.
Figure 6 is a restriction map of plasmid pAHZ108.
Figure 7 is a restriction map of plasmid pMHZ109.

Detailed Description of the Invention In accordance with the present invention, there is provided a DNA fragment containing at least one copy of a first expression cassette comprising in .

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. , ~, : ~ 92/0~1 PCT/~S91/06326 `. 2~8~7~2 the reading frame direction of transcription, the following DNA sequences:
(i) a promoter region of a methanol . responsive gene of a methylotrophic yeast, (ii) a DNA sequence encoding a polypeptide : consisting of:
(a) the S. cerevisiae AMF pre-pro sequence, including the proteolytic i 10 processing site: lys-arg, and ,"! (b) a DNA sequence encoding an human lysozy~e (HLZ) peptide; and (iii) a transcription terminator functional in .. a methylotrophic yeast;
: 15 and optionally further comprising at least one copy of a second expression cassette comprising in the reading frame direction of transcription, the following DNA
sequences:
(i') a promoter region of a methanol responsive gene of a methylotrophic yeast, (ii') a DNA sequence encoding a polypeptide consisting of:
(a') the native human lysozyme signal sequence peptide, and (b') a DNA sequence encoding an human lysozyme peptide; and (iii') a transcription terminator functional in a methylotrophic yeast;
wherein said promoter regions and said transcription terminators are each independently selected from the same or different genes; and wherein said DNA sequences i are operationally associated with one another for '' .
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Wos~/n4~ 14- PCT/US91/063 transcription of the sequences encoding said polypeptide.
The DNA fragment according to the invention can be transformed into methylotrophic yeast cells as a linear fragment flanked by DNA sequences having sufficient homology with a target gene to effect integration of said DNA fragment therein. In this case i integration takes place by addition or replacement at the site of the target gene. Alternatively, the DNA
0 fragment can be part of a circular plasmid, which may be linearized to facilitate integration, and will integrate by addition at a site of homology between the i host and the plasmid sequence.
In accordance with another embodiment of the present invention, there is provided an expression vector containing at least one copy of a DNA fragment as described hereinabove.
i~ According to another aspect of the present invention, there are provided novel methylotrophic yeast cells containing in their genome at least one copy of the above described DNA fragment.
According to a still further embodiment of the present invention, there is provided a process for producing HLZ peptides by growing methylotrophic yeast transformants containing in their genome at least one copy of a DNA sequence operably encoding an HLZ
peptide, operably associated with DNA encoding the S.
cerevisiae AMF pre-pro secretion signal sequence (including the lys-arg proteolytic processing site), and optionally further encoding an HLZ peptide, ; operably associated with DNA encoding the native HLZ
secretion signal peptide, wherein both the coding sequence(s) and the signal sequence(s) are maintained ....
under the regulation of promoter region(s) of methanol ' :, :
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,, ~92/04441 PCT/I,'S91/06326 ., ~8~752 responsive gene(s) of methylotrophic yeast, under conditions allowing the expression of said DNA sequence in said transformants and secreting HLZ peptides into the culture medium. Cultures of viable methylotrophic S yeast cells capable of producing HLZ peptides are also within the scope of the present invention.
The polypeptide product produced in accordance with the present invention is secreted to the culture medium at surprisingly high concentrations;
the level of HLZ peptides secretion mediated solely by the AMF pre-pro secretion signal sequence is more than ten times higher than that obtained with strains wherein secretion is mediated solely by the native lysozyme signal sequence. In addition to the unique properties of the invention expression system, the excellent results obtained in the practice of the present invention are also due to the fact that the S.
cerevisiae alpha-mating factor pre-pro secretion signal sequence (when employed alone, or in combination with the native human lysozyme secretion signal sequence) functions unexpectedly well to direct secretion of HLZ
peptides in methylotrophic yeast.
The term "human lysozyme" or "HLZ peptide" or simply "HLZ", as used throughout the specification and in the claims, refers to a polypeptide product which exhibits similar, in-kind, biological activities to natural human lysozyme, as measured in recognized bioassays, and has substantially the same amino acid sequence as native HLZ. It will be understood that polypeptides deficient in one or more amino acids in the amino acid sequence reported in the literature for naturally occurring HLZ, or polypeptides containing ; additional amino acids or polypeptides in which one or more amino acids in the amino acid sequence of natural .; :.
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HLZ are replaced by other amino acids are within the !' scope of the invention, provided that they exhibit the functional activity of HLZ, e.g., the ability to lyse cells and to produce a stimulatory effect on the phagocytic activity of polymorphonuclear leukocytes and macrophages. The invention is intended to embrace all the allelic variations of HLZ. Moreover, as noted above, derivatives obtained by simple modification of the amino acid sequence of the naturally occurring ; 10 product, e.g, by way of site-directed mutagenesis or other standard procedures, are included within the scope of the present invention. Forms of HLZ produced by proteolysis of host cells that exhibit similar biological activities to mature, naturally occurring HLZ are also encompassed by the present invention.
The amino acids which occur in the various amino acid sequences referred to in the specification have their usual, three- and one-letter abbreviations, routinely used in the art, i.e.:

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~ 92/04~l PCT/~S91/06326 20~9752 Amino Acid Abbreviation L-Alanine Ala A
L-Arginine Arg R
L-Asparagine Asn N
L-Aspartic acid Asp D
L-Cysteine Cys C
L-Glutamine Gln Q
L-Glutamic Acid Glu E
, L-Glycine Gly G
L-Histidine His H
; L-Isoleucine Ile L-Leucine Leu L
L-Lysine Lys K
L-Methionine Met M
L-Phenylalanine Phe F
L-Proline Pro P
L-Serine Ser S
L-Threonine Thr T
L-Tryptophan Trp W
L-Tyrosine Tyr Y
L-Valine Val V
According to the present invention, HLZ
~ peptides are produced by methylotrophic yeast cells ,l 25 containing in their genome at least one copy of a DNA
,' sequence operably encoding HLZ peptides operably "! associated with DNA encoding the S. cerevisiae ~-mating factor ~AMF) pre-pro secretion ~ignal sequonce (including the proteolytic proces~ing slte: lys-arg), and optionally further encoding an HLZ peptide, j operably associated with DNA encoding the native human ~ lysozyme secretion signal peptide, under the regulation ,.~ of promoter region(s) of methanol responsive gene(s) of ~I methylotrophic yeast.
: 35 The term "a DNA sequence operably encoding HLZ peptides" as used herein includes DNA sequences ~ encoding HLZ or any other "HLZ peptide" as defined ,~ hereinabove. DNA sequences encoding HLZ are known in ' the art. They may be obtained by chemical synthesis or , 40 by transcription of messenger RNA (mRNA) corresponding . to HLZ into complementary DNA (cDNA) and converting . :
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the latter into a double stranded cDNA. Chemical synthesis of a gene for HLZ is, for example, disclosed i by Muralli et al., Agric. Biol. Chem., 50: 713-723 (1986); see also Ikehara et al., Chem. Pharm. Bull., 34: 2202 (1986). The requisite DNA sequence can also be removed, for example, by restriction enzyme digest of known vectors harboring the HLZ gene. Examples of such vectors and the means for their preparation are presented in the "Background" section of this disclosure. The structure of a presently preferred HLZ
gene used in accordance with the present invention is illustrated in FIG. 1 and is further elucidated in the examples.
Yeast species contemplated for use in the practice of the present invention are methylotrophs, i.e., species which are able to grow on methanol (as well as other) carbon source nutriment. Species which `~ have the biochemical pathways necessary for methanol utilization fall into four genera, i.e., Candida, j 20 Hansenula, Pichia, and Torulopsis. Of these, a substantial amount is known about the molecular biology of members of the species Hansenula polymorpha and Pichia pastoris.
l The presently preferred yeast species for use ! 25 in the practice of the present invention is Pichia pastoris, a known industrial yeast strain that is capable of efficiently utilizing methanol as the sole carbon and energy source.
There are a number of methanol responsive genes in methylotrophic yeast, the expression of each ` being controlled by methanol responsive regulatory regions (also referred to as promoters). Any of such methanol responsive promoters are suitable for use in , the practice of the present invention. Examples of '':.
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specific regulatory regions include the promoter for the primary alcohol oxidase gene from Pichia pastoris (AOXl), the promoter for the secondary alcohol oxidase gene from P. pastoris (AOX2), the promoter for the dihydroxyacetone synthase gene from P. pastoris (DAS), the promoter for the P40 gene from P. pastoris, the promoter for the catalase gene from P. pastoris, and the like.
The presently preferred promoter region employed to drive HLZ gene expression is derived from a methanol-regulated alcohol oxidase gene of P. pastoris.
P. pastoris is known to contain two functional alcohol oxidase genes: alcohol oxidase I (AOXl) and alcohol oxidase II (AOX2) genes. The coding portions of the two AOX genes are closely homologous at both the DNA
and the predicted amino acid sequence levels and share common restriction sites. The proteins expressed from the two genes have similar enzymatic properties but the promoter of the AOXl gene is more efficient and more highly expressed: therefore, its use is preferred for HLZ expression. The ~Q~ gene, including its promoter, ;` has been isolated and thoroughly characterized; see Ellis et al., Mol. Cell. Biol. 5, 1111 (1985) and US
` 4,855,231.
' 25 The DNA fragment used for transforming methylotrophic yeast cells contains at least one copy of a first expression cassette which, in addition to a methanol responsive promoter of a methylotrophic yeast gene and the HLZ encoding DNA sequence (HLZ gene), contains a DNA sequence encoding, in-reading frame, the S. cerevisiae AMF pre-pro secretion signal sequence, including a DNA sequence encoding the processing site:
lys-arg (also referred to as the lys-arg encoding sequence), and a transcription terminator functional in `'' :
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- a methylotrophic yeast. The DNA fragment used for transforming methylotrophic yeast cells optionally further ~ontains at least one copy of a second expression cassette which, in addition to a methanol responsive promoter of a methylotrophic yeast gene and the HLZ encoding DNA, also includes, in reading frame, the sequence encoding the secretion signal sequence native to HLZ.
The S. cerevisiae alpha-mating factor is a 13-residue peptide, secreted by cells of the "alpha"
mating type, that acts on cells of the opposite "a"
mating type to promote efficient conjugation between the two cell types and thereby formation of "a-alpha"
diploid cells [Thorner et al., The Molecular Bioloqv lS the Yeast Saccharomvces, Cold Spring Harbor Laboratory, Cold Sprlng Harbor, NY, 143 (1981)]. The AMF pre-pro sequence is a leader sequence contained in the AMF
precursor molecule, and includes the lys-arg encoding ,~ sequence which is necessary for proteolytic processing and secretion (see e.g. Brake et al., Proc. Natl. Acad.
Sci. USA, vol. 81, 4642 (1984)). The AMF pre-pro sequence employed in the practice of the present invention is a 255 bp fragment obtained from plasmid pA0208, which is described in W0 89/03907, which is hereby incorporated by reference herein.
The transcription terminator functional in a i methylotrophic yeast used in accordance with the present invention has either (a) a subsegment which encodes a polyadenylation signal and polyadenylation site in the transcript, and/or (b) a subsegment which provides a transcription termination signal for transcription from the promoter used in the expression cassette. The term "expression cassette" as used herein, and throughout the specification and claims, ` ., ': ' ~

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~ 92/0~1 PCT/~'Ssl/06326 .
-21- 2~8~7 refers to a DNA sequence which includes sequences functional for both the expression and the secretion processes. The entire transcription terminator is taken from a protein-encoding gene, which may be the same or different from the gene which is the source of the promoter.
For the practice of the present invention it is preferred that multiple copies of the above-described expression cassettes be contained on one DNA
fragment, preferably in a head-to-tail orientation.
The DNA fragments according to the invention optionally further comprise a selectable marker gene.
For this purpose, any selectable marker gene functional in methylotrophic yeast may be employed, i.e., any gene which confers a phenotype upon methylotrophic yeast cells, thereby allowing them to be identified and ! selectively grown from among a vast majority of untransformed cells. Suitable selectable marker genes include, for example, selectable marker systems composed of an auxotrophic mutant P. pastoris host strain and a wild type biosynthetic gene which complements the host's defect. For transformation of His4 P. pastoris strains, for example, the S.
cerevisiae or P. pastoris HIS4 gene, or for transformation of Arg4 mutants, the S. cerevisiae ARG~
gene or the P. pastoris ARG4 gene, may be employed.
If the yeast host is transformed with a linear DNA fragment containing the HLZ gene under the i regulation of a promoter region of a P. pastoris gene and signal sequences necessary for processing and ~ secretion, the DNA fragment is integrated into the host ; genome by any of the gene replacement techniques known in the art, such as by one-step gene replacement [see e.g., Rothstein, Methods Enzvmol. 101, 202 (1983);

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W092/0~ PCT/US91/0632~
r,~

Cregg et al., Bio/Technoloay 5, 479 (1987); and u.s.
Patent No. 4,882,279~ or by two-step gene replacement ~; methods [see e.g., Scherer and Davis, Proc. Natl. Acad.
sci. USA, 76, 4951 (1979)]. The linear DNA fragment is directed to the desired locus, i.e., to the target gene to be disrupted, by means of flanking DNA sequences having sufficient homology with the target gene to effect integration of the DNA fragment therein. One-step gene disruptions are usually successful if the DNA
to be introduced has as little as 0.2 kb homology with ;; the fragment locus of the target gene; it is however, preferable to maximize the degree of homology for efficiency.
If the DNA fragment according to the invention is contained within, or is an expression vector, e.g., a circular plasmid, one or more copies of the plasmid can be integrated at the same or different loci, by addition to the genome instead of by gene disruption. Linearization of the plasmid by means of a suitable restriction endonuclease facilitates integration.
The term "expression vector", as employed herein, is intended to include vectors capable of expressing DNA sequences contained therein, where such sequences are in operational association with other sequences capable of effecting their expression, i.e., promoter sequences. In general, expression vectors usually used in recombinant DNA technology are often in the form of "plasmids", i.e., circular, double-stranded DNA loops, which in their vector form are not bound to the chromosome. In the present specification the terms "vector" and "plasmid" are used interchangeably.
~owov-r, the invention is intended to include other , ' `, .
: :
: . ., . .

~ 92/04~1 2 0 8 9 7 r 2 PCT/~S91/06326 forms of expression vectors as well, which function equivalently.
In the DNA fragments of the present invention, the segments of the expression cassette(s) are said to be "operationally associated" with one another. ~he DNA sequence encoding HLZ peptides is positioned and oriented functionally with respect to the promoter, the DNA sequence encoding the processing and secretion signal, i.e., the S. cerevisiae ~MF pre-pro sequence (including the DNA sequence encoding theAMF processing-site: lys-arg), or the native HLZ
secretion signal sequence, and the transcription terminator. Thus, the polypeptide encoding seqment is transcribed, under regulation of the promoter region, into a transcript capable of providing, upon translation, the desired polypeptide. Because of the presence of the signal sequence, the expressed HLZ
product is found as a secreted entity in the culture medium. Appropriate reading frame positioning and orientation of the various segments of the expression cas6ette are within the knowledge of persons of ordinary skill in the art; further details are given in the Examples.
The DNA fragment provided by the present invention may include sequences allowing for its replication and selection in bacteria, especially E.
coli. In this way, large quantities of the DNA
;~ fraqment can be produced by replication in bacteria.
Methods of transforming methylotrophic yeast, such as, for example, Pichia pastoris, as well as methods applicable for culturing methylotrophic yeast cells containing in their genome a qene encoding a heterologous protein, are known generally in the art.
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According to the invention, the expression cassettes are transformed into methylotrophic yeast , cells either by the spheroplast technique, described by . Cregg et al., in Mol. Cell. Biol. 5, 3376 (1985) and s U.S. Patent No. 4,879,231; or by the whole-cell lithium chloride yeast transformation system [Ito et al., ~g~ic. Biol. Chem. 48, 341 (1984)], with modification necessary for adaptation to methylotrophic yeast, such as P. pastoris [See U.S. Patent No. 4,929,555~. For the purpose of the present invention, the spheroplast method is preferred.
Positive transformants are characterized by Southern blot analysis [Maniatis et al., Molecular Clonina: A Laboratorv Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA
(1982)] for the site of DNA integration; Northern blot ; analysis [Maniatis, 0~. Cit.; R. S . Zitomer and B.D.
Hall, J. Biol. Chem, ~1, 6320 (1976)] for methanol-responsive HLZ gene expression; and product analysis for the presence of secreted ~LZ peptides in the growth . media.
;, Transformed strains, which are of the desired ~ phenotype and genotype, are grown in fermentors. For ; the large-scale production of recombinant DNA-based ;' 25 products in methylotrophic yeast, a three-stage, high cell-density, fed-batch fermentation system is normally ; the preferred fermentation protocol employed. In the ;~ first, or growth stage, expression hosts are cultured in defined minimal medium with an excess of a non-inducing carbon source (e.g., glycerol). When grown on such carbon sources, heterologous gene expression is completely repressed, which allows the generation of cell mass in the absence of heterologous protein "I
~ expression. Next, a short period of growth under . . ~

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conditions of non-inducing carbon source limitation is allowed. Subsequent to the period of growth under limiting conditions, methanol alone (referred to herein as "methanol fed-batch mode") or a limiting amount of a non-inducing carbon source plus and methanol (referred to herein as "mixed-feed fed-batch mode") are added in the fermentor, inducing the expression of the HLZ gene driven by a methanol responsive promoter. This third stage is the so-called production stage.
The term "culture" means a propagation of cells in a medium conducive to their growth, and all sub-cultures thereof. The term "subculture" refers to a culture of cells grown from cells of another culture (source culture), or any subculture of the source '; 15 culture, regardless of the number of subculturings which have been performed between the subculture of interest and the source culture.
According to a preferred embodiment of the present invention, the heterologous protein expression system used for HLZ production utilizes the promoter derived from the methanol-regulated ~Q~ gene of P.
pastoris, which is very efficiently expressed and tightly regulated. This gene can be the source of the i transcription terminator as well. The presently '~ 25 preferred expression cassette comprises, operationally associated with one another, the P. pastoris AOXl promoter, DNA encoding the S. cerevisiae AMF pre-pro sequence (including the DNA sequence encoding the AMF
processing site: lys-arg), a DNA sequence encoding mature HLZ, and a transcription terminator derived from the P. pastoris AOXl gene. Preferably, two or more of such expression cassettes are contained on one DNA
fragment, in head-to-tail orientation, to yield multiple expression cassettes on a single contiguous '`i ;

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WO92/0~ P~T/US91/063 ., DNA fragment. The presently preferred host cells to be transformed with multiple expression cassettes are P.
pastoris cells having at least one mutation that can be complemented with a marker gene present on a transforming DNA fragment. Preferably His4~ (GS115) or Arg4' (GS190) auxotrophic mutant P. pastoris strains are employed.
The fragment containing one or more expression cassette(s) is inserted into a plasmid containing a marker gene complementing the host's defect. pBR322-based plasmids, e.g., pAO815, are preferred. Insertion of one or more copies of an AMF-based HLZ expression/secretion cassette into parent plasmid pAO815 produces plasmids pAHZ106 and pAHZ108;
while insertion of one copy of an AMF-based expression cassette and one copy of a native HLZ signal sequence based expression cassette produces plasmid pMHZ109.
To develop Mut expression strains of P.
~' pastoris (Mut refers to the methanol-utilization phenotype), the transforming DNA comprising the ~' expression cassette(s) is preferably integrated into ; the host genome by a one-step gene replacement " technique. The expression vector is digested with an appropriate enzyme to yield a linear DNA fragment with ends homologous to the ~Q~l locus by means of the ' flanking homologous sequences. This approach avoids the problems encountered with S. cerevisiae, wherein expression cassettes must be present on multicopy ;'' plasmids to achieve high level of expression. As a result of gene replacement, Mut' strains are obtained.
;' ~n Mut' strains, the AOX1 gene is replaced with the ; expression cassette(s), thus decreasing the strains' ability to utilize methanol. A slow growth rate on methanol is maintained by expression of the AOX2 gene , '~ .
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~'~ 92/0~1 PCT/~IS9l/06326 :,''. 2Q897~2 product. The transformants in which the expression cassette has integrated into the AOX1 locus by site- -directed recombination can be identified by first screening for the presence of the complementing gene.
This is preferably accomplished by growing the cells in media lacking the complementing gene product and identifying those cells which are able to grow by nature of expression of the complementing gene. Next, the selected cells are screened for their Mut phenotype by growing them in the presence of methanol and monitoring their growth rate.
To develop Mut~ HLZ-expressing strains, the fragment comprising one or more expression cassette~s) preferably is integrated into the host genome by ,; 15 transformation of the host with a linearized plasmid , comprising the expression cassette(s). The integration is by addition at a locus or loci having homology with one or more sequences present on the transformation , vector.
~ 20 Positive transformants are characterized by ;;' Southern analysi6 for the site of DNA integration; by Northern analysis for methanol-responsive HLZ gene expression; and by-product analysis for the presence of ;, secreted HLZ peptides in the growth media.
~,~ 25 Methylotrophic yeast strains which have integrated one ~; or multiple copies of an expression cassette at a '~ desired site can be identified by Southern blot ,i analysis. Strains which demonstrate enhanced levels of expression of HLZ may be identified by Northern or : 30 product analysis; however, this characteristic is not "
always easy to detect in shake-flask experiments.
Methylotrophic yeast transformants which are identified to have the desired genotype and phenotype ; are grown in fermentors. It is presently preferred to ~ .
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W092/0~ 9~ PCT/~S91/0632 use the three-step production process described above.
The level of HLZ secreted into the media can be determined by Western blot analysis of the media in parallel with an HLZ s`tandard, using anti-HLZ antisera;
by radioimmunoassay (RIA); by enzyme-inhibitor assay;
. or by HPLC after suitable pretreatment of the medium.
Human lysozyme is purified to apparent homogeneity by ion exchange chromatography. The purification scheme typically employed comprises the following steps. First, fermentor broth is ultrafiltered, the filtrate then loaded directly onto a cationic ion exchange column. The loaded column is then washed sequentially with a series of buffers of increasing pH and varying conductivity (to remove endogenous P. pastoris proteins). Finally the HLZ
product is eluted with a high conductivity buffer, dialyzed and lyophilized.
Because the AMF-based secretion of HLZ
produces not only correctly processed human lysozyme, ; 20 but also produces at least two incorrectly processed ; forms of lyeozyme product, the purification scheme ; employed for the purification of AMF-based, secreted ',,t, HLZ must be capable of separating authentic HLZ from the incorrectly processed forms. It has been found that this can readily be accomplished by making several modifications to the purification scheme described above. First, it has been found that the ultrafiltration step can be eliminated, and cell broth loaded directly onto the cationic ion axchange column.
This alone provides a substantial time savings with respect to the processing time required to prepare purified HLZ.
In order to increase the resolving power of the cationic ion exchange media (e.g., sulphopropyl ; .

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92/~4~1 PCT/~'S91/06326 29- 2~8 9 7~2 ; cellulose, sulphoethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, carboxymethyl sephadex, and the like), the ratio of ion exchange material to fermentation broth is at least doubled, relative to the amount of resin ordinarily employed.
Thus, loading levels in the range of about 10 up to 20 ml of fermentation broth per gram of ion exchange resin are employed. After thorough washing of the loaded resin with a sufficient quantity of solvent having a pH
and conductivity effective to cause elution of endogenous P. pastoris proteins, and thereafter the correctly processed HLZ is selectively eluted with a solvent gradient so as to separate the authentic human lysozyme from the incorrectly processed forms. The thorough washing is typically carried out in a stepwise fashion wherein (1) 1-2 volumes (relative to the volume ~; of ion exchange resin) of buffered media having a conductivity in the range of about 15-25 mMho, and a pH
which is higher than the pH of the fermentation broth (e.g., in the range of about 3.5-5.5): followed by (2) 2-4 volumes of buffered media having a conductivity which is lower than that of the initial wash (e.g., in the range of about 10-15 mMhos and a pH which is higher than that of the initial wash (e.g., in the range of about 5.5-7.0).
The gradient employed for selective elution ; of authentic HLZ from the washed cationic ion exchange resin comprises at least 6 volumes (xelative to the volume of ion exchange resin employed) of a buffered media having a pH in the range of about 7.7 up to 8.2;
wherein a linear gradient is employed starting at a conductivity of about 5-25 mMhos, and increasing to a final conductivity in the range of about 40-60 mMhos.

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WO92/04~ PCT/US91/0632 The invention will now be described in greater detail with reference to the following non-limiting examples.

EXAMPLES
P. pastoris is described herein as a model system for the use of methylotrophic yeast hosts.
Other useful methylotrophic yeasts can be taken from four genera, namely Candida, Hansenula, Pichia and Torulopsis. Equivalent species from them may be used as hosts herein primarily based upon their demonstrated characterization of being supportable for gr~wth and exploitation on methanol as a single carbon nutriment source. See, for example, Gleeson et al., Yeast 4, 1 (1988).

Example 1: Construction of human lysozvme expression vectors The expression vector constructions disclosed in the present application were performed using standard procedures, as described, for example in Maniatis et al., SuDra, and Davis et al., Basic Methods in M~leçu~ BiQ~oaY, Elsevier Science Publishing, 25 Inc., New York (1986).
' ; a. Native human lysozvme sianal seauence:
~RLZ103 pHLZ105 . , .
Plasmid pHLZ100 (described in WO 89/03907), a pUC8 vector containing an almost full length cDNA clone ~ for human lysozyme inserted into the PstI site, was ; used to transform E. coli strain MC1061. The sequence ~ of the cDNA clone, shown in Figure 1, lacked the codons .~' ..
-- ~
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:' WO92/04~1 PCT/US91/06326 ~!
-31- 208~7~2 encoding the first four amino acids of the signal sequence. Transformants were screened by examination of BamHI-HindIII restriction enzyme-digested DNA for the presence of an insert of approximately 570 bp containing an internal PstI site. A colony with the expected restriction pattern was used to prepare plasmid DNA. The 570 bp fragment was cloned into the HindIII-SalI sites of mpl8, yielding plasmid pHLZ101.
Site-directed mutagenesis [Zoller and Smith ' 10 Meth. Enzv~ol. 100:468 (1983)] was used to insert an ; EcoRI site immediately following the translation ; termination codon. The mutagenizing oligonucleotide was of the following sequence:
.
.
,; 15 5'-GCC AGT GCC AAG CTT GAA TTC TTA CAC TCC ACA ACC

The mutagenized clone was sequenced to verify the desired addition of an EcoRI site, and then used for a second site-directed mutagenesis to replace the ~'" 20 four amino-terminal amino acids and to insert an ~coRI
! site immediately preceding the initiation codon. The ; mutagenizing oligonucleotide was of the following sequence:
~t~
; 25 5'-AAG CCC CAG AAC AAT GAG AGC CTT CAT GAA TTC GTC GAC , ';' TCT AGA GGA

The fully mutagenized plasmid was called pHLZ102.
~ After the second mutagenesis was confirmed by j 30 seguencing, the lysozyme gene was isolated on an EcoRI
i;l fragment (500 ng) and separately inserted into the unique EcoRI site of the Pichia pastoris vector pAO804 and pA0815 ~25-50 ng). The construction of pA0804 is described in Example 8. Plasmid pA0815 differs from , ~
., .

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WO 92/04441 ",~, PCT/US91/06326 pAO804 in a single restriction site, BamHI (the HindIII/ClaI/HaeIII site of pAO804 is changed to a BamHI site, providing plasmid pA0815). A restriction map of pAO815 is provided in Figure 2. The ligation mixture was transformed into MC1061 cells and ampR
colonies were selected. Correct plasmid exhibited 2100 and 6180 bp sized bands upon digestion with PstI. The resulting expression vectors, pHLZ103 (derived from parent plasmid pAO804) and pHLZ105 (derived from parent plasmid pAO815), respectively, contain the gene for human lysozyme under the control of the Pichia pastoris AOX1 promoter and regulatory regions as well as the ~OXl transcription termination and polyadenylation - signals. In addition, the vectors include the Pichia ,., ~
; 15 pastoris HIS4 gene used for selection in His hosts, and additional 3' AOX1 sequences used to direct !, , integration into the host genome. These plasmids are shown in Figures 3 and 4, respectively.
; The entire lysozyme gene and approximately 20-25 bases each of the promoter and termination j regions were sequenced in pHLZ103 to verify that no v~l changes had occurred during cloning.
,..
; b. S. cerevisiae alpha-matina factor sianal sequence , ~
i. Sinale CODV vector DAHZ106 , . . .
The aMF pre-pro sequence consists of 89 amino acids which function to direct peptides fused to ~; it through the secretory pathway. The ~MF pre-pro region contains an 83-amino acid signal sequence preceding three processing sites, lys-arg and (glu-ala) 2' which are susceptible to the proteolytic action :

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~ 92/04~1 PCT/US91/06326 _33_ 208~7~
of two specific proteases. Cleavage of the fusion protein at the junction of the ~MF pre-pro and peptide sequences by secretory pathway-localized proteases ; allows the peptide to exit the cell.
The lysozyme gene and native signal sequence DNA were isolated from pHLZ102 (see Example 1) on a SalI-HindIII fragment. This SalI-HindIII fragment .; (1400 ng) was then inserted into an M13 vector (loO ng) which contained, in the EcoRI-SmaI sites, a 2?5 bp EcoRI-HindIII fragment from pA0208 encoding the ~MF
pre-pro region, including the three processing sites.
; Plasmid pA0208 is described in Example 6. The ligation was transformed into JM103 cells and DNA from plaques were analyzed. Correct plasmid exhibited 450 and 7500 bp sized bands upon digestion with HindIII-SalI. Site-directed mutagenesis of the resulting plasmid (pHLZ104) t was used to delete the M13 polylinker, the nucleotides encoding the ~glu-ala)2 processing sites, and the DNA
encoding the native signal sequence, thereby fusing the ; 20 sequence encoding the 83-amino acid pre-pro region and lys-arg processing site directly to the first codon of mature human lysozyme. The oligonucleotide used to perfo~ this mutagenesis was of the following sequence:
!~
.," ~j , !~ 25 :` 5'-GTA TCT TTG GAT AAA AGA AAG GTC TTT GAA AGG TGT
,', After the mutagenesis was confirmed by DNA
sequencing, the fusion gene consisting of the DNA
sequences encoding the ~MF pre-pro region ending in the lys-arg processing site and the mature human lysozyme gene was isolated as an EcoRI fragment (500 ng) and inserted into EcoRI-digested Pichia pastoris vector ' pAO815 (50 ng). The ligation was transformed into ... ~ , ~ : . .
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W092/0~1 PCT/US91/0632 MC1061 cells and amp~ colonies were selected. correct plasmid exhibited 1825, 530, and 6130 bp sized bands upon digestion with PstI. The re-sulting single-copy expression vector, pAHZ106 (Figure 5), contains one copy of the ~MF pre-pro-human lysozyme fusion gene under the transcriptional control of the Pichia pastoris AOX1 promoter and regulatory regions, as well as the ~OX1 transcription termination and polyadenylation signals. The vector also includes the Pichia pastoris HIS4 gene for selection in His- hosts and additional 3' AOXl sequences.
! i The entire ~MF pre-pro-human lysozyme fusion gene and approximately 20 nucleotides each of the ,` promoter and termination regions of pAHZ106 were sequenced to verify that the sequences were not altered ,i during the cloning process.

. ii. Two-~opy vector ~AHZ108 The expression cassette consisting of the AO~1 promoter, ~MF pre-pro-human lysozyme fusion gene, and ~Q~l transcription termination region was isolated from pAHZ106 on a ~g~ mHI fragment (250 ng) and inserted back into the unique ~mHI site of pAHZ106 (25 ng). The ligation was transformed into MC1061 cells and ampR colonies were selected. Correct plasmid exhibited 2400, 3700, and 4235 bp sized bands upon digestion with ~g~ mHI. The resulting vector, pAHZ108 (Figure 6), contains two copies of the expression cassette as tandem-repeat units.
';' ' ., .. . . . . . . .

~ 2/04~1 PCT/US91/06326 2~897~2 iii. Two-co~Y vector containinq the native and ~MF pre-pro sianal seauences: pMHZ109 : ' vector pMHZ109, which contains one copy of the native signal sequence-human lysozyme expression cassette and one copy of the ~MF pre-pro-human lysozyme expression cassette, is shown in Figure 7. The ; expression cassette consisting of the AOXl promoter, ~MF pre-pro-human lysozyme fusion gene, and AOXl , transcription termination region was isolated from pAHZ106 on a Ba~HI-~glII fragment (250 ng) and inserted into the unique ~mHI site of pHLZ105 (25 ng). The ~; ligation was transformed into MC1061 cells and ampR
, 15 colonies were selected. Correct plasmid exhibited '~ 2400, 3500, and 423S bp sized bands upon digestion with ~, BalII-BamHI. Analysis of restriction enzyme digests of s~ pMHZ109 verified that the cassettes were joined as ,7' tandem-repeat units.
.:' 20 `' Example 2: Development of hu~an-ly~Q~y~e-expressin~
, E~ storis strains ,, .
~25 a. Mut~ strains ., .
Expression vectors pHLZ103, pHLZ105, pAHZ106, pAHZ108, and pMHZ109 were used to develop Mut- strains of P. pastoris. The Mut phenotype refers to methanol ~tilization. Mut~ strains utilize methanol in a wild-' type fashion by nature of insertion of the expression cassette into the genome by addition rather than by disruption.
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.:: . , ,,,,;, -W092/0~ PCT/~S91/0632 q~ -36-Mut' strains were developed by integration of the entire expression vector into either the AOXl or HIS4 locus by additive homologous recombination. For site-directed addition of the single copy vector into the AOXl locus, the single copy vectors were digested with SacI, which linearizes the vector within the AOXl promoter region. Similarly, for directed addition into the ~ locus, the multicopy vectors were digested with ~I, which linearizes the plasmid within the HIS4 region. As an additional approach, undigested plasmid was allowed to integrate randomly into the ~1 locus at either the 5' or 3' regions found in the plasmid or into the HIS locus. In each of these additive integrations, the coding region of the AOXl gene was undisturbed.
, Mut' transformants resulting from integration by addition of the expression plasmid, either randomly or by site-direction, were initially screened for ; histidine prototropy. The His' transformants then were analyzed by three Southern blots to verify the site of integration of the plasmid and number of copies integrated. For this, chromosomal DNA was digested with EQRI or BalII. The EcoRI digests were probed with pBR322~based plasmids containing either the AOXl 5' and 3' regions or the Pichia pastoris HIS4 gene. The BalII digest was probed with an oligonucleotide homologous to the human lysozyme gene.
... .
b. Mut strains ; To generate Mut strains, which utilize methanol at a rate slower than that of wild-type strains, plasmid pHLZl03 was digested with BqlII. This liberates an expression cassette comprised of the AOXl "
. .

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`', ~ 92/0~1 PCT/~S91tO6326 ; 37 208~7~2 ,~', promoter region, human lysozyme gene, AOXl ,' transcription termination signals, HIS4 gene for , selection, and AOXl 3' region. Both ends of this -,,' expression cassette contain long sequences which are - .
homologous to the 5' and 3' ends of the AOXl locus.
'' The expression cassette is integrated into the AOXl' locus by a homologous recombination event which results ' in the substitution of the BalII-ended expression ,~ cassette for the AOXl structural gene. Positive ''~ l0 transformants were selected first by their His~
~, phenotype and then by their Mut phenotype, i.e. slow ,' growth on methanol.
,' Transformants resulting from integration of , the BalII fragment of pHLZl03 were initially screened for histidine prototropy and were then analyzed for ', growth on methanol-containing plates. Approximately ~;~ 30% were slow growers, indicative of disruption of the Q~l gene. Eight of these Mut' transformants were analyzed by the Southern blots described above.
c. ,Results ', The following representative strains were chosen for further analysis in one and ten liter ,',; 25 fermentations:
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Fragmont 8ite of Phenotype Copy 8trnin Nam~ Inteqri~ted Inte~rntion (Mut +/-~ Number ....
G+HLzloss3 . StuI pHLZlO5 HIS4 +
5 G+AHZlO6S7 StuI pAHZ106 HIS4 +
: G+AHZlO8S20 StuI pAHzlos HIS4 + 2 G+MHzloss2s StuI pMHZ109 HIS4 + (1 each)2 G-HLZlO3S5 BqlII pHLZl03 AOXl - 1 G+HLZl03Sl6 StuI pHLZl03 HIS4 + 3 Example 3: Growth of human lysozvme-expressinq strains in one and ten liter fermentations Media employed in fermentations described herein had the following compositions:
.-' ~
;,' A. lOx ~ASA~ SALTS
Chemical Grams/liter Phosphoric acid, 85% 42.0 ml Calcium Sulfate.2H20 l.8 Potassium Sulfate 28.6 Magnesium Sulfate.7H20 23.4 ' 2S Potassium Hydroxide 6.5 .

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,.
, El. Pl'Ml TRACE SALTS
Chemical Grams/liter Cupric Sulfate.5H20 6.0 Sodium Iodide 0.08 ; 5 Manganese Sulfate.H20 3.0 Sodium Molybdate.2H20 0.2 Boric Acid 0.02 Cobalt Chloride 0.5 Zinc Chloride 20.0 Ferrous Sulfate.7H20 65.0 Biotin 0.20 Sulfuric Acid s.o ml a. One-liter fermentations: Mut+ strains ; Run 686 G+MHZ109S25 (one copy of native signal ; sequence-human lysozyme gene and one copy of ~MF pre-pro-;l human lysozyme fusion gene) ~'l 20 ;;~ Run 687 G~AHZ108S20 (two copies of ~MF pre-pro-human lysozyme fusion gene) ::, Run 688 G+AHZ106S~ (one copy of ~MF pre-pro-human lysozyme fusion gene) .,.,~
Run 689 G+HLZ105S3 (one copy of native signal sequence-human lysozyme gene) ;;~ 30 The fermentor was autoclaved with 1000 ml of medium containing 500 ml of 10X basal salts, 5%
4j glycerol, and the remainder deionized water. After sterilization, 4 ml of PTM1 trace salts were added, and the pH was adjusted to 5.0 with concentrated NH~OH.

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; 40-The pH of the medium was maintained at 5 by addition of 50% NH40H containing 0.1% Struktol J-673 antifoam.
Inocula were prepared from buffered YNB containing 2%
glycerol. The fermentor was inoculated with 10-50 ml of the cultured cells which had grown to an OD~o of 1-- 8, and the batch growth regimen was continued for 18-24 hours. At the point of glycerol exhaustion, as indicated by increased dissolved oxygen, a glycerol feed (50% glycerol plus 12 ml/L PTMl salts) was initiated at 5-20 ml/hour and continued until 200 ml of glycerol feed had been added. Subsequently, the glycerol feed was terminated, and a methanol feed (100%
methanol plus 12 ml/L PTM1 salts) was started at an initial rate of approximately 2 ml/hour. After 2 hours, the methanol feed rate was increased in in~rements of 10% every 30 minutes until a final rate of 5-5.5 ml/hour was attained. The vessel was harvested 70-100 hours following methanol induction.
Results are summarized below:
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WO92/0~ PCT/US91/063 As shown in the preceding Table, the cell - growth of the four strains were similar and the fermentations reached fi~al wet cell weights of approximately 350-420 g/L. This level of cell density is typical for Pichia pastoris strains grown under the standard Mut~ conditions. The similar amount of growth of the strains containing one and two copies of the ~MF
pre-pro-human lysozyme fusion gene expression cassette ; 10 indicates that the presence of additional copies of the cassette does not adversely affect cell growth.
The levels of human lysozyme secreted by these strains during one-liter fermentations were measured by both enzyme activity assay (Example 5.c) and RIA (Example 5.d). The human lysozyme expression level data for the fermentations are included in the Table set forth above. Strain G+HLZ105S3, which contains one copy of the native signal sequence-human lysozyme gene expression cassette, secreted only 17 mg of human lysozyme/L after 94 hours of induction by methanol. The strain containing one copy of the ~MF
pre-pro-human lysozyme fusion gene expression cassette, G+AHZ106S7, secreted approximately 200 mg/L (as determined by enzyme activity assay of the broth). The strain containing two copies of the same expression cassette, G+AHZ108S20, secreted greater than 600 mg/L.
Strain G+MHZ109S25, which contains one copy of each of the expression cassettes, secreted slightly more human lysozyme than G+AHZ106S7 (285 mg/L versus 200 mg/L), but less than G+AHZ108S20.
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~ ~92/~4~l PCT/~S9l/06326 2~897.~2 b. One-liter fermentations: Mut- strains ;; Run 518 G-HLZl03S5 (one copy of native signal sequence-human lysozyme gene) Inocula were prepared from selective plates and grown overnight at 30~C in buffered YNB containing 2% glycerol to an OD~o of 1-8. An aliquot of 5-50 ml of the overnight culture was added to a 2-liter capacity fermentor, and the repressed growth phase ;; continued in 5X basal salts containing 5 ml/L of PTM
salts at 30DC. The pH was maintained at 5.5 by the addition of 50% ~v/v) ammonium hydroxide, and foaming was controlled by the addition of 5% ~v/v) Struktol antifoam. Dissolved oxygen was maintained above 20% by increased aeration and agitation as needed. This batch growth phase continued for 18-24 hours until the glycerol was exhausted. Following exhaustion of the ' glycerol in the initial growth phase, a 50% (w/v) glycerol feed (containing 12 ml/L of PTMl trace salts) was initiated at a rate of 5-20 ~l/hour. After the additlon Or approximately 100 ml of 50% glycerol, a methanol feed (100% plus 12 ml/L of PTMl trace salts) , was inltiated at a rate of l ml/hour. The rate was increased over the course of the fermentation to maintain a residual methanol concentration of less than 0.5~. The vessel was harvested 70-150 hours following , methanol induction.
; The average yield of human lysozyme for strain G-HLZ103S5 was approximately 10 mg/L of cell-free fermentor broth (enzyme activity).
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9~a c. Ten-liter fermentations: Mut~ strains .
i. Standard Mut' protocol .: .
Run 660 G+HLZ103S16 ; Run 695 G+AHZ108S20 A 15-liter capacity fermentor was autoclaved with 6.5 liters of a solution containing 5.5 liters of lOX basal salts and 525 g of glycerol. After sterilization, the pH was adjusted to 5.0 with NH3 gas, and 30 ml of PTM1 trace salts were added. The ~;; fermentor was inoculated with 500 ml of an overnight culture grown in buffered YNB containing 2% glycerol.
The pH was maintained at 5.0 by the addition of NH3 gas, and the temperature was maintained at 30 C. A 5%
solution of Struktol J-673 was added as necessary to control foaming. Dissolved oxygen was maintained above ; 20% saturation by increased agitation and aeration as needed.
At the point of glycerol exhaustion, indicated by increased dissolved oxygen, a glycerol ; feed ~50% glycerol plus 12 ml/L of PTMl salts) was initiated at 50-200 ml/hour and continued until a volume of 700 ml had been added. Following this glycerol fed-batch phase, a methanol feed (100%
; methanol plus 12 ml/L PTM1 salts) was initiated at 7.5-10 ml/hour. After two hours at this rate, the methanol ` feed was increased by 10% increments every 30 minutes to a final rate of 60 ml/hour which was maintained for the remainder of the fermentation.
In Run 660, the cell density reached 500 g/L
after 92 hours on methanol, and approximately 24 mg of human lysozyme were secreted per liter of fermentation .,'' - .
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W092/04~1 PCT/~S91/06326 2~8~7~2 broth. In Run 695, the cell density reached 452 g/L
after 72 hours on methanol, and approximately 670 mg of human lysozyme were secreted per liter. These levels of secreted lysozyme are similar to those achieved in the one-liter fermentation of these strains and indicate that strain G+AHZ108S20 and G+HLZ103S16 can be grown in large-scale fermentations with no loss of productivity.

'4'~ 10 ii. Increased methanol feed protocol Run 704 G+AHZl08S20 Strain G+AHZ108S20, containing two ~MF pre-pro-human lysozyme fusion gene cassettes, was also grown in a 10-liter fermentation conducted using an increased methanol feed during the induction phase.
The first two phases of Run 704 were conducted according to the standard conditions described above.
Following the glycerol fed-batch phase, the methanol feed was initiated at 20 ml/hour. After 3 hours, the feed rate was increased by 20% increments every 15 minutes to a final rate of 60 ml/hour.
The cell yield (final cell density of 459 g/L) was similar to the density achieved in the previous 10-liter fermentation of this strain (Run 695); and, although slightly less, the yield of human lysozyme (550 mg/L) was similar. Therefore, it appears that either protocol can be used for 10-liter fermentations of this strain.
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d. Ten-liter fermentations: Mut strains ;~ Run 667 G-HLZ103S5 (1 copy) , "
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WO92/0~ PCT/US91/06326 Mut- strain G-HLZl03S5 was grown in a lO-liter fermentation according to a standard Mut protocol. The initial two phases of Run 667 were conducted as described for the standard Mut~ lO-liter fermentation protocol. In the induction phase, the methanol feed was introduced at 7.5-lO ml/hour and increased after two hours by 10% increments every 30 minutes until a final rate of 30 ml/hour was achieved.
At this rate of methanol addition, the residual methanol level in the fermentor was maintained between O.l and 0.5%.
Under the standard Mut conditions, strain G-HLZl03S5 grew to a final cell density of 428 g wet weight/L, which is typical of other Mut recombinant strains grown in lO-liter fermentations. The level of ; human lysozyme produced in Run 667 was approximately 20 mg/L-" , Example 4: Purification of secreted human lvsozvme ; 20 a. ~rification of human 1YSOZYme secreted usina native sianal ~eouence ,' In general, the purification scheme consisted of four basic steps. First, fermentor broth was filtered through a lO0,000 molecular-weight cut-off spiral cartridge. Second, the filtrate, which contained the human lysozyme, was loaded directly onto a radial flow sulphopropyl cartridge. In the third step, the cartridge was washed sequentially with a series of buffers with increasing pH and varying conductivity to remove endogenous Pichia pastoris proteins. Finally, the human lysozyme was eluted from ' ' . .
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This procedure was applied to purify human lysozyme produced in the standard lO-liter Mut~
s fermentation of the three-copy strain G+HLZl03S16 (Run 660 Example 3.c.i) ! which was harvested after 92 hours of growth on methanol. Cell-free broth, obtained by centrifugation of the contents of the fermentor at 6500 X g for 30 minutes, was stored frozen at -20~C.
The broth was thawed at 4-C overnight and at 37'C
immediately prior to purification. All subsequent steps of the purification were carried out at 4 C. The broth ~approximately 5050 ml) was pumped, at a rate sufficient to produce 20 psi back pressure, through a 100,000 molecular-weight cut-off spiral cartridge (Amicon Spiral Ultrafiltration Cartridge, type SlY100).
After approximately 4.5 liters had been filtered, one liter of 50 mM sodium acetate, pH 5.1, containing sufficient NaCl to bring the conductivity of the solution to 24.7 mMho/cm2, which is similar to the ; conductivity of fermentation broth, was added to the reservoir and filtered. Approximately 5.4 liters of filtrate were recovered from the cartridge and 0.7 liters of retentate remained.
A 250-ml ZetaPrep Modular radial flow sulphopropyl cartridge (Cuno Inc., Meriden, CT) was activated as recommended by the manufacturer. The filtrate from the spiral ultrafiltration cartridge was pumped directly onto the sulphopropyl cartridge at 5 - 30 ml/minute. The cartridge was then washed with a series of six buffers of increasing pH and conductivity as follows:

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W092/04~1 ~9~ PCT~S91/0632 1. 1 liter of 50 mM sodium acetate, pH 5.1, containing sufficient NaCl to bring the - conductivity to 24.7 mMho/cm2, . ~ .
2. o.S liters of 50 mM sodium acetate, pH
5.1, 8.9 mMho/cm2, 3. 0.25 liters of 50 mM sodium phosphate buffer, pH 7.0, 6.8 mMho/cm2, '' 10 4. 0.5 liters of 50 mM sodium phosphate, pH
7.0, containing sufficient NaCl to bring the conductivity to 14.8 mMho/cmZ, :

5. 0.25 liters of 50 mM sodium phosphate, pH 7.0, 6.8 mMho/cm2, .~ . .

6. 0.5 liters of 50 mM sodlum phosphate, pH
8.0, 7.3 mMho/cm2.
; Following the washes, the human lysozyme was -, eluted from the sulphopropyl cartridge with one liter of 50 mM sodium phosphate, pH 8.0, containing sufficient NaCl to bring the conductivity to ~ 25 approximately 100 mMho/cm2. Fractions of approximately ,' 30 ml were collected, and the absorbance of the fractions was measured at 280 nM. The peak fractions containing material with absorbance at 280 nM were pooled and dialyzed in Spectrapor tubing with a ';i~ 30 molecular-weight cut-off of 6000-8000 against two liters of MilliQ-purified water. The dialysis was changed every four hours for two days. The sample was ;^ then frozen and lyophilized.
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WO 92/04441 PCTtl 'S9 1 /06326 ~,11 , ~,," 2~897~2 An outline of the purification scheme and the yields of human lysozyme at each step, as determined by RIA, are presented in Table 2. Approximately 82% (89 mg) of the 109 mg of human lysozyme in the starting , 5 cell-free broth was recovered from the 100,000 molecular-weight cut-off spiral cartridge. This material was applied to a ZetaPrep cartridge and then eluted off of the cartridge as a single peak with the buffer of high conductivity. In order to ensure a product with the greatest purity, only fractions from the major portion of the peak were pooled. This sample contained approximately 74 mg of human lysozyme, a 68 recovery of the starting material. Extensive characterization of this material revealed that the human lysozyme had been correctly processed and was at least 95% pure according to analysis by SDS-PAGE, immunoblot, amino acid analysis, and/or protein sequencing (see Example 5.e-h). The later fractions of , the peak were also pooled. RIA analysis of this pool , 20 indicated that it contained an additional 16 mg (14% of ,i the starting material) of human lysozyme. Thus, the , total recovery was approximately 82~ of the starting ", material and 100~ of the lysozyme applied to the ZetaPrep cartridge.

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WO9Z/~Jl~9~ PCI/US91/0632 . -50-TABT.T'. 2 . PURIFICATION OF HUMAN LYSOZYME PP~ODUCED BY
: STRAIN G+HLZ103S16 HUMAN TOTAL HUMAN
sVOLUME LYSOZYME LYSOZYME PERCENT
PURIFICATION STEP (ml)(~g/ml) (mg) RECOVERY
Cell-free broth 505021.5 108.6 100 Fermentor Run 660 100,000 MWCO Spiral Cartridge Eluate 540016.5 89.1 82 Retentate 700 37.6 26.3 24 Zeta Prep Cartridge ; Filtrate 5000 0.8 4.0 3.6 . 15 Wash 1:
.; 50 mM Na Acetate ~ pH 5.1, 24 mMho/cm2 1000 0.2 .02 <0.1 ,~. Wash 2:
'. 50 mM Na Acetate pH 5.1, 8.9 mMho/cmZ 500 .94 0.47 .4 , Wash 3:
'~ 50 mM Na Phosphate pH 7.0, 6.8 mMho/cm2 2S0 .01 0.002 ~0.1 Wash 4:
50 mM Na Phosphate pH 7.0,14.8 mMho/cmZ 500 .01 0.005 <0.1 Wash 5: <0.1 ,~ 50 mM Na Phosphate ;' pH 7.0, 6.8 mMho/cmZ 250 .01 0.002 ,' 30 Wash 6:
. 50 mM Na Phosphate pH 8.0, 7.3 mMho/cm2 500 .01 0.005 <0.1 Eluate:
i, 50 mM Na Phosphate ;,, 35 pH 8.0, 100 mMho/cmZ
, Pool 1: 155 476 73.8 68 Pool 2: 180 87.4 15.7 14 .:
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b. Purification of human lysozvme secreted usinq S. cerevisiae alDha-matin~ factor sianal seouence Because several different forms of human lysozyme were present in the fermentation media of strains containing the AMF pre-pro secretion signal sequence, the scheme used to purify the human lysozyme produced by strain G+HLZ103S16 was extensively modified for purification of the lysozyme produced by these strains. The major form, which comprised about 66% of , the total, is authentic human lysozyme. The remaining forms comprise human lysozyme having an N-terminal extension of 9 or 11 amino acids (from the C-terminal region of pre-pro AMF).
Two sulphopropyl cartridges were used in series to accommodate the increased level of human lysozyme in the fermentation broth. Secondly, elution of the human lysozyme from the sulphopropyl cartridges was achieved with a gradient system developed to separate the ma~or forms of the protein produced by the ¦ new strains. In addition, other modifications were " .
, introduced to simplify the overall purification :' procedure. Filtration of the cell-free broth through the 100,000 molecular-weight cut-off spiral cartridge was eliminated, and the nu~ber of washes of the ZetaPrep cartridges was reduced.
This modified scheme was used to purify the ;i; human lysozyme produced in ten-liter fermentation Run ; 30 704 of strain G~AHZl08S20. The contents of the fermentor were harvested after 70 hours on methanol and centrifuged at 6500 X g for 30 minutes. The cell-free broth (6100 ml) was pumped at 5 ml/minute directly onto the two ZetaPrep Modular radial flow sulphopropyl '"":

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WO92/0~ PCT/US91/06326 cartridges connected in series. The cartridges were washed with 0.8 liters of 50 mM sodium acetate, pH 5.0, containing sufficient NaCl to bring the conductivity to 20 mMho/cm2, followed by l.4 liters of 50 mM sodium phosphate, pH 7.0, with sufficient NaCl to bring the conductivity to 13.8 mMho/cm2.
The human lysozyme was eluted from the cartridges with a four-liter linear gradient of 50 mM
sodium phosphate, pH 8.0, with increasing conductivity ; 10 from lO to 50 mMho/cm2. Fractions of approximately lO
ml were collected and the absorbance of the fractions was measured at 280 nM. Aliquots of fractions containing material with absorbance at 280 nM were . analyzed by tricine SDS-PAGE to monitor the elution of the two forms of lysozyme produced by strain G+AHZl08S20. Fractions containing only the correctly processed human lysozyme, as determined by gel electrophoresis and protein sequencing, were pooled and dialyzed in Spectrapor tubing with a molecular-weight cut-off of 6000-8000 against continuously running deionized water for 24 hours. The dialyzed material was then ~rozen and lyophilized.
' An outline of the purification scheme and the yields of human lysozyme at each step, as determined by RIA, are presented in Table 3. Following centrifugation, the cell-free broth from fermentation Run 704 was sufficiently clear to pump directly onto the ZetaPrep cartridges. (If the cell-free broth is stored at -20-C before purification, it might be ; 30 necessary to include filtration through the lO0,000 molecular-weight cut-off spiral cartridge before binding onto the ZetaPrep cartridges. Broth which has ; been frozen frequently becomes more viscus and contains insoluble mate~ial which can easily clog the ZetaPrep .
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92/O~ CT/US91/06326 cartridge.) In order to ensure the highest purity, the fractions ~#281-340) corresponding to the region of the absorbance peak were pooled (Eluate Pool l, Table 3).
This pool contained approximately l gram of human lysozyme which appeared to be correctly processed and at least 95% pure according to analysis by SDS-PAGE, immunoblot, amino acid analysis, and/or protein sequencing (see Example 5.e-h). The fractions comprising the other region of the peak, fractions 261-280, were also pooled and characterized. This pool (Eluate Pool 2, Table 3) contained an additional 694 mg of human lysozyme which also appeared to be correctly processed according to analysis by SDS-PAGE, immunoblot, amino acid analysis, and/or protein sequencing (see Example 5.e-h).
; The cell-free broth contained approximately 2.8 g of human lysozyme immunoreactive material, as determined by RIA, of which approximately 1.9 g (66~) ! was estimated to be correctly processed. As shown in Table 3, approximately 1.7 grams (or 61~) of the 2.8 grams of immunoreactive starting material was isolated.
However, the 2.8 grams o~ starting material contained ~ both correctly and incorrectly processed human ; lysozyme, approximately 1.9 and 0.9 grams, respectively. Thus, the approximately 60% recovery noted in Table 3 represents a recovery of approximately 89% of the correctly processed human lysozyme.

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TABT~ 3 , PURIFICATION OF HUMAN LYSOZYME PRODUCED BY
:: STRAIN G+AHZ108S20 TOTAL HUMAN HUMAN

PURIFICATION STEP (ml) (~g/ml) (mg) RECOVERY
Cell-Free Fermentor Broth Run 704 6100 458 2793 100 ZetaPrep Cartridge Filtrate 6000 S.2 31.2 <0.1 Wash 1:
. 50 mM Na Acetate, ~: pH 5.0, 20.0 mMho/cm2 800 6.6 5.3 <0.1 Wash 2:
50 mM Na Phosphate ' pH 7.0, 13.8 mMho/cm2 1400 0.8 1.1 <0.1 '. Eluate:
50 mM Na Phosphate .
,: pH 8.0, 10 mMho-50 ,. 20 mMho/cm Pool 1:
Fractions 281-340 770 3569 987.0 33.0 ,,: Pool 2:
r~" Fractions 2~1-280 200 3470 694.0 24.8 ,, 25 . .
Example 5: Assays The amounts of human lysozyme secreted from recombinant strains of Pichia pastoris were quantitated by RIA and lysis assays of cell-free broth obtained during growth of the strains in fermentors.

:', a. Standard human lysozyme and Dolvclonal ~ 35 antisera .~ , Human lysozyme from two sources was used as standard in RIA, immunoblot, and enzyme activity , .. ..

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~ 2/0~W1 PCT/US91/06326 208~7a2 assays. Lysozyme from the urine of human leukemia patients was purchased from Green cross corporation (distributed by Alpha Therapeutic Corporation, Los Angeles, CA), and lysozyme isolated from human milk was obtained from Sigma Chemical Company (St. Louis, MO).
The two human lysozymes were compared in RIA and enzyme activity assays and found to be identical within ~ experimental error. Concentrations oP stock solutions f were determined spectrophotometrically from the extinction coefficient, E2~ (1%, l cm) = 25.5.
Polyclonal rabbit antisera to human lysozyme, purchased from Dako-Immunoglobulins (Santa Barbara, CA), were used for the RIA and immunoblot analyses.
,.',' ' ;i 15 b. PreDaration of cell-free broth and cell extracts of recombinant Pichia Dastoris cells Samples from fermentor cultures of human lysozyme-expressing strains of Pichia pastoris were aentrifuged at 6500 X g for 5 minutes to separate the ; broth and the cells. The broth was decanted from the cell pellet and used for immunoblot, RIA, and enzyme activity assays.
Cell extracts were prepared from 175 mg of cells grown in fermentors. The cells were washed twice in extraction buffer (10 mM sodium phosphate, pH 7.5, 0.1% Triton X-100, 0.5 M NaCl, 2 mM PMSF), and then vortexed four times for 1 minute each with 0.5 g of 0.5 mm glass beads and 0.35 ml of extraction buffer. The beads were washed with an additional 0.35 ml of extraction buffer which was combined with the lysate ` and centrifuged in a microfuge for 15 minutes. The supernatant was removed, and the total protein was :

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W092/04~l PCT/~S91/06326 9~

determined by the Bradford method [Anal. Biochem.
72:248 (1976)]. The remaining cell pellets were suspended in 0.7 ml of 2X sample buffer (0.125 M Tris HCl, pH 6.8, 4% SDS, 200 mM dithiothreitol (DTT), 20%
glycerol, 0.005% bromophenol blue, 20 ~g/ml pyronin Y), boiled for 10 minutes, and centrifuged in a microfuge for 15 minutes. Following centrifugation, the supernatant was removed and retained. The cell lysates and supernatants resulting from centrifugation of resuspended insoluble pellets were analyzed by immunoblot.

c. Enzvme activitv assay The lysis activity of the human lysozyme produced in Pichia pastoris was measured, with modifications, as described [Shugar et al. Biochem.
Biophvs. Acta 8:302 (1952)]. Lysozyme was added to a suspension of Mic~ococcus lysodeikticus, and the decrease in absorbance at 405 nm was measured spectrophotometrically. The standard conditions used for the assay were pH 7.4 in 0.033M potassium phosphate buffer containing 0.1% BSA at 25'C. In a typical assay, 1 ml of Micrococcus lysodeikticus suspended at a concentration of 0.1 mg/ml in 0.033 M potassium phosphate, pH 7.4 containing 0.1% BSA, was pipetted into a one-ml plastic cuvette and the OD405 measured. A
0.05-0.25 ~g (in a volume of 25 ~1 or less) aliquot of standard human lysozyme or fermentor broth (diluted as necessary in 0.033 M potassium phosphate, pH 7.4 containing 0.1% BSA) was added at time zero. Following the addition of standard or sample, the absorbance at OD40s was recorded for two minutes. The specific activity of the standard human lysozyme is defined as ;

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-57- 20~9752 the change in OD405 per minute per mg of lysozyme. The average specific activity of both the milk and urine ; lysozymes was found to be ~ 1100 OD405/minute/mg in the enzyme activity assay under the standard conditions.
The concentration of human lysozyme in the fermentation ; broth was calculated from the specific activity obtained for the standard in the same assay.
In order to verify that the enzyme activity assay is valid for crude broth samples, human milk lysozyme was assayed in the presence of cell-free broth from fermentor runs of human lysozyme-expressing strains. The broth had no effect on the activity of the standard, i.e. the activities of the standard lysozyme in the broth and the Pichia pastoris-produced lysozyme in the broth were additive. This result indicates that cell-free broth does not interfere with the assay.

d. RIA
A radioimmunoassay (RIA) was developed for the evaluation of the expression of human lysozy~e.
Iodinated human lysozyme required for the RIA was not commercially available, therefore, lysozyme isolated from the urine of human leukemia patients was iodinated by an Iodobead procedure. For this, 50 ~g of lysozyme was combined with one Iodobead (Pierce, Rockford, IL) and 1 mCi of [125I]NaI (NEN, Boston, MA) in 50 mM NaPO4, pH 7.4. After incubation for 30 minutes on ice, the l25I-lysozyme was separated from free iodine on a Sephadex G-25 column. The material collected in the peak fraction appeared to be 95~ intact as determined ; by tr1chloroacetic acid precipitaticn.

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W092/0~ PCT/US9l/0632 With a standard RIA protocol, there was an unacceptable level of non-specific binding of the iodinated lysozyme which resulted in a high background.
Incubation conditions were studied in order to reduce the background, and it was determined that the non-specific binding was due to adsorption of the labeled lysozyme to Pansorbin. Therefore the concentration of Pansorbin was optimized to a level which does not compromise the signal, but does produce a significantly lower background.
In the optimized protocol for the human lysozyme RIA, approximately 12,000 cpm of 125~-lysozyme were incubated overnight at 4'C with polyclonal antisera (final dilution of 1:25,000) and varying amounts of unlabeled standard lysozyme or broth samples. The binding reaction was carried out in glass tubes in a final volume of 0.5 ml containing 50 mM
sodium phosphate, pH 7.5/O.l M NaCl/25 mM EDTA/0.1%
NaNJO.l% BSA (fraction V)/0.1% Triton X-lO0.
Following the incubation, lO0 ~l of Pansorbin (working dilution of 1:320; Calbiochem, San Diego, CA) was added, and the tubes were incubated for 15 minutes at ; room temperature. Two ml of wash buffer (0.9% NaCl/5 mM EDTA/0.1% Trlton X-lO0) were added, and the tubes were centrifuged at 3200 RPM for 68 minutes at 4 C (J6M
centrifuge), decanted, and the radioactivity in each pellet was counted. Total binding in the absence of unlabeled lysozyme was approximately 32% while non-specific binding was less than 6% of the total counts of the iodinated lysozyme. The EDso for the competition of the 125I-lysozyme by unlabeled lysozyme was 2.5 ng, and the sensitivity of the assay was approximately 0.8 ng. The standard curves generated in .
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~92tO4~1 PCT/~S91/06326 ,, .~,, -5~-the RIA of the human lysozymes purified from milk and urine are superimposable.

e. SDS-PAGE

SDS-PAGE analyses were performed essentially as described b~ Laemmli [Nature 227:680 (1970)]. Each sample was diluted 1:1 in 2X sample buffer (0.125 M
Tris HCl, pH 6.8, 4% SDS, 200 mM DTT, 20% glycerol, 0.005% bromophenol blue, 20 ~g/ml pyronin Y) and boiled for 10 minutes before separation in a Mini-Protein gel apparatus (BioRad, Richmond, CA) on either a 15%
acrylamide gel with a 5% stacking gel or a 17-27%
gradient Sepragel precast gel (Integrated Separation Systems, Hyde Park, MA). Protein standards (BioRad) were included as molecular weight markers.
Alternatively, SDS-PAGE analyses were performed in a tricine system as described by Schagger and von Jagow [_nal. Biochem. 166:368 (1987)~ on a 16%
acrylamide gel with a 4% stacking gel. The 2X sample buffer for the tricine system consisted of 8% SDS, 24%
glycerol, 0.1 M Tris HCl, pH 6.8, 200 mM DTT, 0.004%
Coomassie Brilliant Blue G.
Gels were stained with Coomassie Brilliant Blue R followed by silver. For Coomassie staining, gels were stained overnight in 50% ethanol, 10% acetic acid, 5% TCA, 200 mg/L Coomassie Brilliant Blue. The ; following day, the gels were rehydrated for one hour in 10% ethanol, 10% acetic acid, 1% TCA, 50 mg/L Coomassie Brilliant Blue, and then destained in 10% ethanol, 10%
acetic acid. Silver staining was accomplished essentially as described by Morrissey [Anal. Biochem.
:; 117:307 (1981)] without the glutaraldehyde fixation.

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Wo 92/04441 pcr/us91/o6326 i; c~ -60-v~:~ f. Immunoblots , ~ , .
Cell extracts and cell-free broth samples -~ analyzed by immunoblot were first separated by SDS-PAGE, as described above. The gels were not subjected to staining. Transfer of protein to 0.45 : nitrocellulose was carried out for 1.5 hours at o.1 i amps. The membrane was blocked with western buffer ~, (0.25% gelatin, lX PBS, 0.05% Tween-20, 0.02% sodium azide) at 37-C for 1 hour before incubation overnight at room temperature in Western buffer containing a 1:2000 dilution of polyclonal antisera to lysozyme ; isolated from human urine. The membrane was then washed extensively (four 15-minute washes) with Western ;, 15 buffer, incubated with approximately 3 ~Ci 1Z5I-Protein ~ A (New England Nuclear, Boston, MA) at room temperature ;, for 60 minutes, washed extensively as before, air ~ dried, and exposed to film. Under these conditions, as ....
~ little as 1 ng of human lysozyme can be detected.
;~ 20 g. Am~nQ acid analvs~g ."",~ .
Purified human lysozyme was subjected to amino acid analysis following hydrolysis [Spackman et al. ~1958). Anal. Biochem. 30:1190.~ A known ~ amount of purified human lysozyme was added to a glass ol tube, and the tube was placed in a reaction flask containing approx~mately 0.5 ml of 6N HCl. After evacuation, the sample was hydrolyzed by vapor phase hydrolysis at llO-C for 24 hours. The hydrolyzed sample was then taken up in 500 ~l of 0.2 M sodium ~¦ acetate, pH 2.2, and 50 ~il were applied to a ~eckman 6300 Amino Acid Analyzer.

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WO92/04~l PCT/~'S91/06326 ~.
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-61- 208 9 7 ~2 h. Protein sequencina , The N-terminal amino acid sequence of samples of purified Pichia pastoris-produced human lysozyme was determined according to the method of Hunkapiller and Hood [Science 219:650 (1983)]. An Applied Biosystems 470/120 Gas Phase Protein Sequencer was utilized in the analysis of -l nmol of purified material.
., ', Example 6: Construction of ~lasmid ~AO208 The AOX1 transcription terminator was isolated from 20 ~g of pPG2.0 [pPG2.0 = BamHI-HindIII
fragment of pG4.0 (NRRL 15868) + pBR322] by StuI
digestion followed by the addition of 0.2 ~g SalI
linkers (GGTCGACC). The plasmid was subsequently ; digested with HindIII and the 350 bp fragment isolated from a 10% acrylamide gel and subcloned into pUCl8 (Boehringer Mannheim) digested with HindIII and SalI.
The ligation mix was transformed into JMl03 cells (that , are widely available) and ampR colonies were selected.
The correct construction was verlfled by HindIII and SalI digestion, which ylelded a 350 bp fragment, and ,' was called pA0201.
5 ~g of pA0201 was digested with HindIII, : filled in using E. coli DNA Polymerase I Klenow , ., ~ fragment, and 0.1 ~g of BglII linkers (GAGATCTC) were ,~ added. After digestion of the excess 8glII linkers, the plasmid was reclosed and transformed into MCl061 cells. Amp~ cells were selected, DNA was prepared, and the correct plasmid was verified by BglII, SalI double digests, yielding a 350 bp fragment, and by a HindIII
digest to show loss of HindIII site. This plasmid was called pAO202.

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W092/04l~ PCI/US91/063 An alpha factor-GRF fusion was isolated as a 360 bp BamHI-PstI partial digest from pYSV201. Plasmid pYSV201 is the EcoRI-BamHI fragment of GRF-E-3 inserted into M13mpl8 (New England Biolabs). Plasmid GRF-E-3 is described in European Patent Application No. 206,783.
20 ~g of pYSV201 plasmid was digested with BamHI and partially digested with PstI. To this partial digest was added the following oligonucleotides:
., , 5' AATTCGATGAGATTTCCTTCAATTTTTACTGCA 3' 3' GCTACTCTAAAGGAAGTTAAAAATG 5'.

only the antisense strand of the oligonucleotide was kinase labelled so that the oligonucleotides did not polymerize at the 5'- end. After acrylamide gel electrophoresis (10%), the fragment of 385 bp was isolated by electroelution. This EcoRI- BamHI fragment of 385 bp was cloned into pA0202 which had been cut with EcoRI and BamHI. Routinely, 5 ng of vector cut with the appropriate enzymes and treated with calf : intestine alkaline phosphatase, was ligated with 50 ng of the insert fragment. MC1061 cells were transformed, ampr cells were seleCted, and DNA was prepared. In this case, the resulting plasmid, pA0203, was cut with EcoRI and BglII to yield a fragment of greater than 700 bp. The ~-factor-GRF fragment codes for the (1-40)1eu27 version of GRF and contains the processing sites lys-arg-glu-ala-glu-ala.
The AOXl promoter was isolated as a 1900 bp EcoRI fragment from 20 ~g of pAOP3 and subcloned into EcoRI-digested pA0203. The development of pAOP3 is disclosed in European Patent Application No. 226,846 and described hereinbelow. MC1061 cells were transformed with the ligation reaction, amp' colonies . .
.

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were selected, and DNA was prepared. The correct orientation contains a ~376 bp HindIII fragment, whereas the wrong orientation has an ~675 bp fragment.
One such transformant was isolated and was called pA0204.
The parent vector for pA0208 is the HIS4, PARS2 plasmid pYJ32 (NRRL B-15891) which was modified to change the EcoRV site in the tet~ gene to a BglII
site, by digesting PYJ32 with EcoRV and adding BglII
linkers to create pYJ32(+BglII). This plasmid was digested with BglII and the 1.75 Kb BglII fragment from pA0204 containing the AOXl promoter-~ mating factor-GRF-AOX1 3' expression cassette was inserted. The rèsulting vector was called pA0208. An EcoRI digest of pAO208 yielded an 850 bp~fragment + vector, while vector having the other orientation yielded a 1.1 Kb fragment + vector.

Construction of ~lasmid DAOP3:
1. Plasmid pPG2.5 [a pBR322 based plasmid containing the approximately 2.5 Xbp EcoRI-SalI
fragment from plasmid pPG4.0, which plasmid contains the primary alcohol oxidase gene (AOX1) and regulatory regions and which is available in an E. coli host from the Northern Regional Research Center of the United States Department of Agriculture in Peoria, Illinois as NRRL B-15868] was linearized with BamHI.
2. The linearized plasmid was digested with BAL31:
; 3. The resulting DNA was treated with E.
coli DNA Polymerase I Klenow fragment to enhance blunt ends, and ligated to EcoRI linkers;
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WO92/0~ PCT/US91/0632 4. The ligation products were transformed into E. coli strain MM294;
5. Transformants were screened by the !, colony hybridization technique using a synthetic oligonucleotide having the following sequence:
5'-TTATTCGAAACGGGAATTCC-3'.
This oligonucleotide contains the AOX1 promoter sequence up to, but not including, the ATG initiation codon, fused to the sequence of the EcoRI linker;
6. Positive clones were sequenced by the Maxam-Gilbert technique. All three positives had the ; following sequence:
5'...TTATTCGAAACG_GGAATTCC...3'.
They all retained the "A" of the ATG (underlined in the above sequence). It was decided that this A would probably not be detrimental; thus all subsequent clones are derivatives of these positive clones. These clones have been given the laboratory designation pAOP1, pAOP2 and pAOP3, respectively.
Example 7: Construction of ~la~mid pA0815 , Plasmid pA0815 was constructed by mutagenizing plasmid pA0807 (which was in turn prepared as described hereinbelow) to change the HindIII/ClaI/
HindIII sites downstream of the AOXl transcription ; terminator in pA0807 to a BamHI site. The oligonucleotide used for mutagenizing pA0807 had the following sequence:
5'-GAC GTT CGT TTG TGC GGA TCC AAT GCG GTA GTT TAT-3'.

The mutagenized plasmid was called pA0807-Bam. Plasmid pA0804 was digested with BglII and 25 ng of the 2400 bp : .
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WO92/04~1 PCT/~S91/06326 , .;~. .
; 208~7~2 fragment were ligated to 250 ng of the 5400 bp BglII
;~ fragment from sglII-digested pAo807-sam. The ligation mix was transformed into MC1061 cells and the correct - construct was verified by digestion with Pst/BamHI to identify 6100 and 2100 bp sized bands. The correct construct was called pA0815. The restriction map of the expression vector pAO815 is shown in Figure 3.

Construction of ~lasmid DA0807:
: 10 l. Preparation of fl-ori DNA:
fl bacteriophage DNA (50 ~g) was di~ested with 50 units of Rsa I and Dra I (according to manufacturer's directions) to release the ~458 bp DNA fraqment containing the fl origin of replication (ori). The digestion mixture was extracted with an equal volume of phenol: chloroform (V/V) followed by extracting the aqueous layer with an equal volume of chloroform and finally the DNA in the aqueous phase was precipitated by ! 20 adjusting the NaCl concentration to 0.2M and adding 2.5 ,'; volumes of absolute ethanol. The mixture was allowed to stand on ice (4-C) for 10 minutes and the DNA precipitate was collected by centrifugation for 30 minutes at 10,000 ' x g in a microfuge at 4-C.
The DNA pellet was washed 2 times with 70~
aqueous ethanol. The washed pellet was vacuum dried and `, dissolved in 25 ~l of TE buffer [1.0 mM EDTA in 0.01 M
(pH 7.4) Tris buffer]. This DNA was electrophoresed on 1.5% agarose gel and the gel portion containing the ~458 ; 30 bp fl-ori fragment was excised out and the DNA in the gel was electroeluted onto DE81 (Whatman) paper and eluted ' from the paper in lM NaCl. The DNA solution was precipitated as detailed above and the DNA precipitate ,~ was dissolved in 25 ~l of TE buffer (fl-ori fragment).

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WO92/04~1 PCT/~IS91/06326 q~
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~8 -66-2. Cloning of fl-ori into Dra I sites of pBR322:
pBR322 (2 ~g) was partially digested with 2 units Dra I (according to manufacturer's instructions).
The reaction was terminated by phenol:chloroform extraction followed by precipitation of DNA as detailed ; in step 1 above. The DNA pellet was dissolved in 20 ~1 of TE buffer. About 100 ng of this DNA was ligated with 100 ng of fl-ori fragment (step 1) in 20 ~1 of ligation buffer by incubating at 14-C for overnight with 1 unit of T4 DNA ligase. The ligation was terminated by heating to 70'C for 10 minutes and then used to transform E. coli strain JM103 [Janisch-Perron et al., Gene, vol 22, 103(1983)]. AmpR transformants were pooled and superinfected with helper phage R408 [Russel et al., supra]. Single stranded phage were isolated from the media and used to reinfect JM103. AmpR transformants contained pBRfl-ori which contains fl-ori cloned into the ~, Dra I sites ~nucleotide positions 3232 and 3251~ of pBR322.
, ', 3. Construction of plasmid pA0807:
' pBR~l-ori (10 ~g) was digested for 4 hours at ~, 37'C with 10 units each of Pst I and Nde I. The digested DNA was phenol:chloroform extracted, precipitated and dissolved in 25 ~1 of TE buffer as detailed in step 1 , above. This material was electrophoresed on a 1.2%
, agarose gel and the Nde I - Pst I fragment (approximately 0.8 kb) containing the fl-ori was isolated and dissolved ; 30 in 20 ~1 of TE bufrer as detailed in step 1 above. About 100 ng of this DNA was mixed with 100 ng of pA0804 (which ', was in turn prepared as described hereinbelow) that had been digested with Pst I and Nde I and phosphatase-treated. This mixture was ligated in 20 ~1 of ligation ;

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W092/0~ PCT/~'S91/0632h 20897~2 ~

buffer by incubating overnight at 14~C with l unit of T4 DNA ligase. The ligation reaction was terminated by heating at 70C ~or 10 minutes. This DNA was used to transform E. coli strain JMl03 to obtain pA0807.
Example 8: Construction of plasmid ~A0804:

Plasmid pA0804 has been described in PCT
Application No. WO 89/04320. Construction of this plasmid involved the following steps:
Plasmid pBR322 was modified as follows to eliminate the EcoRI site and insert a BglII site into the PvuII site:
, pBR322 was digested with EcoRI, the protruding ends were filled in with Klenow Fragment of E. coli DNA
polymerase I, and the resulting DNA was recircularized using T4 ligase. The recircularized DNA was used to transform E. coli MC1061 to ampicillin-resistance and transformants were screened for having a plasmid of about 4.37 kbp in size without an EcoRI site. One such transformant was selected and cultured to yield a plasmid, designated pBR322~RI, which i8 pBR322 with the EcoRI slte replaced with the sequence:
,. .
: `~ 25 51 -GAATTAATTC-3 ' ,.
3 '-CTTAATTAAG-5 ' .

,~ pBR322~RI was digested with PvuII, and the linker having the sequence:
5 ' -CAGATCTG-3 ' 3 ' -GTCTAGAC-5 ' , .,,. :

W092/04~1 PCT/~S9l/0632~
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w ~ligated to the resulting blunt ends employing T4 ligase. The resulting DNAs were recircularized, also with T4 ligase, and then digested with sglII and again recircularized using T4 ligase to eliminate multiple ; 5 BglII sites due to ligation of more than one linker to the PvuII-cleaved pBR322~RI. The DNAs, treated to eliminate multiple BglII sites, were used to transform E.
coli MC1061 to ampicillin-resistance. Transformants were screened for a plasmid of about 4.38 kbp with a BglII
site. One such transformant was selected and cultured to yield a plasmid, designated pBR322~RIBGL, for further work. Plasmid pBR322~RIBGL is the same as pBR322~RI
except that pBR322~RIBGL has the sequence , . . .
5'-CAGCAGATCTGCTG-3' 3'-GTCGTCTAGACGAC-5' -: .
; in place of the PvuII site in pBR322~RI.
ii pBR322ARI8GL was digested with a SalI and BglII
and the large fragment ~approximately 2.97 kbp) was isolated. Plasmid pBSAGISI, which i5 described in ~4i European Patent Application Publicat$on No. 0 226 752, was digested completely with BglII and XhoI and an approximately 850 bp fragment from a region of the P.
,~, 25 pastoris AOXl locus downstream from the AOX1 gene , transcription terminator (relative to the direction of transcription from the AOXl promoter) was isolated. The BglII-XhoI fragment from pBSAGISI and the approximately 2.97 kbp, SalI-BglII fragment from pBR322~RIBGL were ~;1 30 combined and subjected to ligation with T4 ligase. The ligation mixture was used to transform E. coli MC1061 to ~ ampicillin-resistance and transformants were screened for : a plasmid of the expected size (approximately 3.8 kbp) with a BglII site. ~his plasmid was designated pA0801.

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The overhanging end of the SalI site from the pBR322~RIBGL fragment was ligated to the overhanging end of the XhoI site on the 850 bp pBSAGI5I fragment and, in the process, both the SalI site and the XhoI site in pA0801 were eliminated.
pBSAGI5I was then digested with ClaI and the approximately 2.0 kbp fragment was isolated. The 2.o kbp fragment has an approximately 1.0-kbp segment which comprises the P. pastoris AOXl promoter and transcription initiation site, an approximately 700 bp segment encoding the hepatitis B virus surface antigen ("HBsAg") and an approximately 300 bp segment which comprises the P.
pastoris AOX1 gene polyadenylation signal and site-encoding segments and transcription terminator. The HBsAg coding segment of the 2.0 kbp fragment is terminated, at the end adjacent the 1.0 kbp segment with the AOX1 promoter, with an EcoRI site and, at the end ;, adjacent the 300 bp segment with the AOXl transcription terminator, with a StuI site, and has its subsegment which codes for HBsAg oriented and positioned, with respect to the 1.0 kbp promoter-contalning and 300 bp transcription terminator-containing segments, operatively for expression of the HBsAg upon transcription from the ' AOXl promoter. The EcoRI site joining the promoter ,~ 25 segment to the HBsAg coding segment occurs just upstream (with respect to the direction of transcription from the ; AOX1 promoter) from the translation initiation signal-encoding triplet of the AOXl promoter.
; For more details on the promoter and terminator segments of the 2.0 kbp, ClaI~site-terminated fragment of ~ pBSAGI5I, see European Patent Application ~ublication No.
,~ 226,846 and Ellis et al., Mol. Cell Biol. 5, 1111 (1985).
Plasmid pA0801 was cut with ClaI and combined for ligation using T4 ligase with the approximately 2.0 :
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~ -70-kbp ClaI-site-terminated fragment from pBSAGI5I. The ligation mixture was used to transform E. coli MC1061 to ampicillin resistance, and transformants were screened for a plasmid of the expected size (approximately 5.8 kbp) which, on digestion with ClaI and BglII, yielded fragments of about 2.32 kbp (with the origin of replication and ampicillin-resistance gene from pBR322) and about 1.9 kbp, 1.48 kbp, and 100 bp. On digestion with BglII and EcoRI, the plasmid yielded an approximately 2.48 kbp fragment with the 300 bp terminator segment from the AOXl gene and the HBsAg coding segment, a fragment of about 900 bp containing the segment from upstream of the AOXl protein encoding segment of the AOX1 gene in the AOXl locus, and a fragment of about 2.42 kbp containing the origin of replication and ampicillin resistance gene from p~R322 and an approximately 100 bp ClaI-BglII segment of the .:.
AOX1 locus (further upstream from the AOXl-encoding segment than the first mentioned 900 bp EcoRI-BglII
segment). Such a plasmid had the ClaI fragment from pBSAGI5I in the desired orientation, in the opposite undesired orientation, there would be EcoRI-BglII
fragments of about 3.3 kbp, 2.38 kbp and 900 bp.
One of the transformants harboring the desired plasmid, designated pA0802, was selected for further work and was cultured to yield that plasmid. The desired orientation of the ClaI fragment from pBSAGI5I in pA0802 had the AOXl gene in the AOXl locus oriented correctly to lead to the correct integration into the P. pastoris genome at the AOXl locus of linearized plasmid made by cutting at the BglII site at the terminus of the 800 bp fragment from downstream of the AOXl gene in the AOX1 1 ocus .

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~ ~ 2/~41 PCT/~S91/0632( .,,,. 208~7~2 pA0802 was then treated to remove the HBsAg coding segment terminated with an EcoRI site and a StuI
site. The plasmid was digested with StuI and a linker of sequence:

5'-GGAATTCC-3' 3'-CCTTAAGG-5' .
was ligated to the blunt ends using T4 ligase. The mixture was then treated with EcoRI and again subjected to ligating using T4 ligase. The ligation mixture was then used to transform E. coli MC1061 to ampicillin resistance and transformants were screened for a plasmid of the expected size (5.1 kbp) with EcoRI-BglII fragments : 15 of about 1.78 kbp, 900 bp, and 2.42 kbp and BglII-ClaI
fragment of about 100 bp, 2.32 kbp, 1.48 kbp, and 1.2 kbp. This plasmid was designated pA0803. A transformant with the desired plasmid was selected for further work and was cultured to yield pA0803.
Plasmid pA0804 was then made from pA0803 by inserting, into the BamHI site from pBR322 in pA0803, an approximately 2.75 kbp BglII fragment from the P.
pastoris ~IS4 gene. See, e.g., Cregg et al., Mol. Cell.
~iol. 5, 3376 (1985) and European Patent Application Publication Nos 180,899 and 188,677. pA0803 was digested with BamHI and combined with the HIS4 gene-containing BglII site-terminated fragment and the mixture subjected to ligation using T4 ligase. The ligation mixture was used to transform E. coli MC1061 to ampicillin-resistance and transformants were screened for a plasmid of the expected size (7.85 kbp), which is cut by SalI. One such transformant was selected for further work, and the plasmid it harbors was designated pA0804.

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. ~o9 pA0804 has one SalI-ClaI fragment of about l.5 kbp and another of abut 5.0 kbp and a ClaI-ClaI fragment of l.3 kbp; this indicates that the direction of : transcription of the HIS4 gene in the plasmid is the same ; 5 as the direction of transcription of the ampicillin resistance gene and opposite the direction of ; transcription from the AOXl promoter.
The orientation of the HIS4 gene in pA0804 is ' not critical to the function of the plasmid or of its derivatives with cDNA coding segments inserted at the .~~ EcoRI site between the AOXl promoter and terminator '; segments. Thus, a plasmid with the HIS4 gene in the orientation opposite that of the HIS4 gene in pA0804 would also be effective for use in accordance with the present invention.
~i The invention has been described in detail with ;~ reference to particular embodiments thereof. It will be understood, however, that variations and modifications can be effected within the spirit and scope of the ; 20 invention.

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Claims (28)

WHAT IS CLAIMED IS:

1. A DNA fragment comprising at least one copy of a first expression cassette, wherein said expression cassette comprises, in the direction of transcription, the following DNA sequences:
(i) a promoter region of a methanol responsive gene of a methylotrophic yeast, (ii) a DNA sequence encoding a polypeptide consisting of:
(a) the S. cerevisiae AMF pre-pro secretion signal sequence, including the processing site:
lys-arg, and (b) a DNA sequence encoding an human lysozyme (HLZ) peptide: and (iii) a transcription terminator functional in a methylotrophic yeast;
and optionally further comprising at least one copy of a second expression cassette comprising in the reading frame direction of transcription, the following DNA
sequences:
(i') a promoter region of a methanol responsive gene of a methylotrophic yeast, (ii') a DNA sequence encoding a polypeptide consisting of:
(a') the native human lysozyme signal sequence peptide, and (b') a DNA sequence encoding an human lysozyme peptide; and (iii') a transcription terminator functional in a methylotrophic yeast;
wherein said promoter regions and said transcription terminators are each independently selected from the same or different genes; and wherein said DNA sequences are operationally associated with one another for transcription of the sequences encoding said polypeptide.

2. A DNA fragment according to Claim 1 further comprising at least one selectable marker gene and a bacterial origin of replication.

3. A DNA fragment according to Claim 2 wherein said fragment is contained within a circular plasmid.

4. A DNA fragment according to Claim 1 wherein said sequence encoding an HLZ peptide has the sequence set forth in Figure 1.

5. A DNA fragment according to Claim 1 wherein said methylotrophic yeast is a strain of Pichia pastoris.

6. A DNA fragment according to Claim 5 wherein said methanol responsive gene of a methylotrophic yeast and the transcription terminator are both derived from the P. pastoris AOX1 gene.

7. A DNA fragment according to Claim 6 further comprising 3'- and 5'-ends having sufficient homology with a target gene of a yeast host for said DNA

fragment to effect site-directed integration of said fragment into said target gene.

8. A DNA fragment according to Claim 1 further comprising 3'- and 5'-ends having sufficient homology with a target gene of a yeast host for said DNA
fragment to effect site-directed integration of said fragment into said target gene.

9. A DNA fragment according to Claim 1 containing multiple copies of said first expression cassette.

10. A DNA fragment according to Claim 9 wherein said multiple copies of said first expression cassette are oriented in head-to-tail orientation.

11. A DNA fragment according to Claim 1 containing at least one copy of said first expression cassette and at least one copy of said second expression cassette.

12. A DNA fragment according to Claim 7, which is derived from a SacI digest of the Pichia expression vector pALZ106 or a StuI digest of the Pichia expression vector pALZ108 or pMHZ109.

13. A methylotrophic yeast cell transformed with the DNA fragment of Claim 1.

14. A methylotrophic yeast cell according to claim 13 wherein said yeast is a strain of Pichia pastoris.

15. A methylotrophic yeast cell transformed with the DNA fragment of Claim 4.

16. A methylotrophic yeast cell transformed with the DNA fragment of Claim 9.

17. A P. pastoris cell transformed with the DNA fragment of Claim 7.

18. A P. pastoris cell according to Claim 17, wherein said cell is selected from strain G+AHZ106S7, G+AHZ108S20, G+AHZ109S25, or G+MHZ109S25.

19. A culture of viable methylotrophic yeast cells according to Claim 13.

20. A culture of viable P. pastoris cells according to Claim 17.

21. A culture of visible P. pastoris cells according to Claim 18.

22. A process for producing human lysozyme (HLZ), said process comprising growing the cells of Claim 13 under conditions allowing the expression of said expression cassette(s) in said cells, and the secretion of said HLZ product into the culture medium.

23. A process according to Claim 22 wherein said methylotrophic yeast is a strain of Pichia pastoris.

24. A process according to Claim 22 wherein said cells are grown in a medium containing methanol as a carbon source.

25. A process according to Claim 22 wherein said cells have the Mut+ phenotype.

26. A process according to Claim 22 wherein said cells have the Mut? phenotype.

27. A method for purifying a mixture of human lysozyme and N-terminal extended homologs thereof, said method comprising:
(a) contacting cationic ion exchange resin with in the range of about 10-20 ml of the fermentation broth obtained from the process of Claim 22 per gram of resin;
(b) washing the resulting HLZ-loaded resin with a sufficient quantity of solvent having pH and conductivity suitable to cause elution of endogenous P. pastoris proteins; and thereafter (c) selectively eluting correctly processed HLZ with a solvent gradient.

28. In a method for purifying recombinantly produced human lysozyme by ion exchange chromatography, the improvement comprising applying the fermentation broth obtained from the process of Claim 22 directly to cationic ion exchange resin at a loading level in the range of 10-20 ml of fermentation broth per gram of resin, washing the resulting HLZ-loaded resin with a sufficient quantity of solvent having pH and conductivity suitable to cause elution of endogenous P. pastoris proteins; and thereafter selectively eluting correctly processed HLZ with a solvent gradient.