NL8202851A - EXPRESSION VECTORS. - Google Patents

Description

y - ί) VO 3556 », i 1.

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, Expression vectors.

In the genetic conversion technique, a common technique for causing a foreign protein to be produced by a procariotic organism is a transformation of this organism with a plasmid that has been modified to contain a genetic information speci fi c for desired foreign protein. Various eucariotic genes have been successfully inserted into plasmids and, after insertion into procariotic organisms, have been used successfully to produce these foreign proteins by these organisms. For example, a production of human interferon, human growth hormone, human insulin, chicken ovalbumin, somatostatin and the like has been reported with genetically modified strains of Escherichia coli, ie Proc. Hatl. Acad. Sci. U.S.A., 77, 5230 - 5233 (1980); ibidem 75, 3727-3731 (1978) and Nature 281, 544 (1979).

Constructing an organism capable of producing a foreign protein 15 involves a number of steps. First, the gene carrying the instructions for the biosynthesis of the desired protein must be identified and isolated. Methods for this identification and isolation of genes vary widely, but any method according to which the desired gene can be obtained in a substantially pure form can be used.

A method commonly used to obtain eucariotic genes begins with the isolation of messenger RM (mRM). Protein synthesis is initiated in cells by a transcription comprising an enzymatic synthesis of an EM chain corresponding to the DNA template. The RITA thus formed interacts with the cell's protein synthesizing device to produce the protein. The RM, specified by a specific gene (thus also specifying a special protein) is referred to as messenger RM. When the cell produces the protein, this mRM is present at much higher concentrations than the gene carrying the protein synthesizing information. In addition, the mRM is usually in a much more suitable form for isolation and purification than chromosome DM. Usual purification techniques ,. such as density gradient sedimentation, 8202851

i V

2 affinity chromatography and electrophoresis can be used to isolate mRNA from cell lysates or homogenates. These techniques have already been used, for example, for the fins of insulin mRNA from pancreatic cells and interferpn mRNA from human leukocytes as well as fibroblast cells.

The mRNA thus obtained can then be used to make a single stranded DNA molecule {known as complementary DNA (cDM) see Science 196; 1313 (1977)). The resulting single stranded DNA molecule has a 3 'terminal hairpin structure which provides a primer for a subsequent synthesis of a second strand of DNA using the enzyme DNA polymerase. The loop connecting the two strands of DNA is then disrupted (e.g. with the enzyme S1 nuclease) resulting in a cDNA segment identical to the original gene. The resulting cDNA is usually isolated and purified, for example, by a gel electrophoresis and its structure can be verified by a sequence analysis or by the restriction method. The ends of DNA segments thus obtained can be modified as described below by facilitating insertion into a transfer vector.

Genes can be inserted into procariotic cells via transfer vectors, which are conventionally plasmids or bacteriophages, into which the gene of interest is inserted. When the purpose of inserting the gene into an organism is to multiply this gene, i.e. useful amounts of this gene by obtaining clones, the transfer vector is conventionally referred to as "cloning vector". When the purpose of inserting the gene into an organism is to produce a protein, the transfer vector is sometimes referred to as an "expression vector". The present invention relates to new transfer vectors and, in particular, plasmid expression vectors. Plasmids are relatively small closed loops of DNA. An improved understanding of the activity of restriction endonuclease enzymes and the discovery and isolation of some of these enzymes have enabled molecular biologists to characterize and modify plasmids for improved utility as transfer vectors . For example, undisturbed DNA can be spiked, reducing the size of the plasmid; restriction sites can be made ready 8202851 ¥. - * 3 mq.fl.irh for gene insertion "and selection promoting genes, such as antibiotic resistance, can be inserted into a plasmid.

Simply pasting a gene into a plasmid does not guarantee protein production. Protein synthesis requires the presence of various control signals in the vicinity of the gene (Aie Cell, 20, 5, 3-553 (1980). These signals initiate, regulate and terminate, for example, an mRNA transcription as a mRNA translation (i.e., an egg protein synthesis). It has further been found that control signals associated with a bacterium synthesis of a specified egg protein may be used to control the bacterial protein synthesis of a foreign protein. has been advantageously used to construct widely applicable plasmid extrusion vectors Czie Eroc, Natl, Acad.

Sci. II.S.A j6; T60 - 4 (19T9) -

The control signals associated with protein synthesis are graphically shown in Figure 1 of the drawing, which depicts a segment of double-stranded DNA, associated with control of the enzymes involved in tryptophan biosynthesis. "p'r gives a base pair order spring, which is known as the promoter region. During protein synthesis, the promoter region signals the initiation of the 20 mRNA transcription." 0 "gives a base pair order spring which is known is a sales region This region, together with a repressor, controls the abundant mRNA transcription For example, in a tryptophan biosynthesis, an excess of tryptophan enters into fisheries breeding with a repressor, thereby decreasing the amount of trp-mRNA transcription. promoter-25 and sales regions are shown as both pieces, some acquisition regions may be components within a promoter sequence. "SD" represents a base pair sequence spring, known as the Sbine-Dalgarno sequence. is the signal by the egg-synthesizing device (the ribosome) recognizes the mRNA, so the Sbine-Dalgarno sequence is not needed for an mRNA synthesis but sheet for a protein synthesis. "AIG" gives a methionine or initiator- codon spring. This codon is the signal for the initiation of an egg-synthesis in the bacterium.

These signals are largely idiosyncratic, meaning that different organisms apply different codes. Therefore, if a eucariotic gene containing eucariotic regulatory regions is introduced into a plasmid and subsequently inserted into a procariotic cell, this cell may produce 8202851 $ k geea protein or even mMA because it cannot recognize the required control sequences. On the other hand, a promoter region for the synthesis of a specific protein in a procariotic organism can be combined with a eucariotic gene and use a plasmid clone with such a combination of the protein-synthesizing device of such a procariotic organism to make the foreign protein.

Molecular biologists have taken advantage of this discovery to synthesize so-called portable promoters, which portable promoters are shown in Figure 2 and contain a premotor, a working and a Shine-Dalgarno region, which can be recognized by a specific host. -organism. They are bound at each end by specific restriction enzyme recognition sequences, which allow them to be excised and inserted elsewhere after. an appropriate enzymatic treatment. Preferably, the opposite ends of such a portable promoter have base pair sequences corresponding to different restriction enzymes. In Figure 2, these ends are designated X and Y, corresponding to various restriction enzymes, which are arbitrarily designated X and Y. Such a structure ensures proper orientation of the portable promoter toward the gene to be expressed; and it also provides a single insertion site in the resulting plasmid (as further explained below). A plasmid as shown in Figure 3 with restriction sites X and Y corresponding to each end of the portable promoter can be opened by decomposition with appropriate restriction enzymes and the portable promoter can then be inserted therein, for example using the enzyme ligase. The resulting plasmid (Figure b) can then be used as an expression vector.

To use such an expression vector, the desired gene is isolated as described above and ends are chemically modified to have sequences corresponding to the Y restriction enzyme. If the gene lacks initiation and termination codons, each end must be modified to contain such codons. After opening the restriction enzyme Y plasmid, the modified gene can be inserted to form the desired plasmid (Figure 5). Transformation of an appropriate procariotic organism with such a plasmid can result in expression of the foreign 8202851 S egg protein under control of the tryptofan control signals. Free skin any bacterium promoter-sales-Sbine-Dalgarno region can be used for this purpose.

As set forth above, genes inserted in hitherto known transfer vectors must contain initiation and termination signals. In the case of termination signals, this requirement is not particularly difficult because with isolation of bet. gene. A restriction enzyme can be selected such that the termination codon is included, and there are many difficulties if vender DM (after the termination codon) is also obtained.

On the other hand, the space between the Shine-Dalgarno sequence and the initiation codon can be very important. To find a restriction enzyme ^ that a gene from a. cutting out a longer DM strand in such a way that the initiation codon is obtained without also obtaining unduly long "leader" pieces of DM is very complicated. Taker 15 is cut off the gene such that the initiation codon and part of the gene. lose. The lost DM, the initiation codon and the appropriate leader must usually be added enzymatic or chemical spring.

The invention now relates to a new expression vector for introducing a gene into a procariotic organism. This expression vector comprises a closed loop of DM with signals that allow it to replicate in a procariotic host boar cell, and includes verier (a) promoter, sales, and translation initiation sequences, which can be recognized by the procariotic host boar cell; (B) a Shine-Dalgarno sequence and an initiator codon within the translation initiation sequence; and (c) a restriction endo-nuclease recognition sequence located near the initiator codon and leaving this initiator codon or its complement within the translation initiation sequence upon cleavage.

The new expression vector of the invention is shown in Figure 6 of the drawing. Figure 6 shows a cireular plasmid spring with promoter, sales and Shine-Dalgarno regions and the methionine (ATG) initiator codon. A site for a restriction endonuclease 35 (randomly designated Z) is near the initiator codon. Such a transfer vector can be used very widely because on opening 8202851 6 of. a plasmid with restriction endonuclease Z and insertion of a gene, the plasmid has all the signals necessary for an mRNA transcription and for a translation of a gene encoding the desired foreign protein.

In the plasmid expression vector of the invention, the composition and length of the segment between the Shine-Dalgarno sequence and the initiator codon can be accurately and reproducibly controlled. Thus, this segment can be made ready for the specific promoter work system used, for optimal gene expression. In addition, the expression vector has an insertion site positioned such that cleavage of the plasmid with the appropriate restriction endonuclease leaves the initiator codon in the translation initiation sequence. Thus, manipulation of the expression vector to insert a gene does not disrupt the translation initiation sequence, so that this sequence remains intact regardless of the gene to be inserted. Here, the "translation initiation sequence" includes the Shine-Dalgarno sequence - (ribosome binding plates), the initiator codon (normally ATG) and the DBA segment between these two sequences.

The expression vectors of the invention can be obtained from natural plasmids or modifications thereof.

Preferably, the plastoid replication functions used as the starting material do not have any impairment of subsequent operations. These replication functions ensure that the final transfer vector is subject to the desired mode of replication control, ie, it is contained in a number of copies or a single copy per cell or in an adjustable number of copies per cell. Such a plasmid starting material is also preferably relatively small in size. Because the plasmid is small, it can accept large genes as shear, cell transformation is facilitated, and the plasmid does not unnecessarily divert large amounts of cell energy and nutrients into the production of unwanted macromolecules. Preferably, the plasmid should be less than about 10 kilobases, preferably between two kilobases to about 5 kilobases. To prepare an expression vector of the invention, control signals associated with RKA transcription and translation are inserted into the starting plasmid. Although such control signals may be in any form transmitted by the desired guest. 82 0 2 8 5 1 '----------------------------- -------------- "7 lord organism are recognized, certain systems are preferred. The promoter-working regions that control procariotic protein synthesis vary in effectiveness. Usually highly effective system is preferred because such a system results in higher protein production. Known promoter-work systems which are particularly useful for synthesizing the new transfer vectors of the invention are the Escheriehia systems for controlling the lactose (.lac) and tryptophan (trp) metabolism and the promoter working region (P1) of the baeterio phage X. The lac and trp systems are usually preferred for the synthesis of the subject expression vectors.

These control signals can be obtained by cutting out any DM containing them. The cut-out method depends on the restriction enzymes available and the presence of useful restriction sites that hold the system together. The promoter, work and Shine-Delgarno regions are preferably recovered as an intact segment DM. The excised DM segment with the desired control signals is then inserted into the plasmid using conventional gene insertion methods.

Also, a plasmid that already contains the desired control 20 signals can be used as a starting material for synthesizing the subject expression vectors. A particularly favorable plasmid starting material is referred to as pGLIO1 and has been described by Guarente et al. (See above); this plasmid graphically depicted in Figure 7 contains restriction sites for each of the enzymes EcoRI and EcvuII, and carries genes for ampicillin resistance. This plasmid also contains the promoter-work-Shine-Dalgarno sequence of an E. coli lac operon. The system was obtained from a lac UY5 mutant by decomposition with Alul restriction endonuclease. The UV5 mutation renders the lac promoter insensitive to catabolic suppression by catabolic gene activator protein 30 (CAP). The Alul ends of the promoter-work-Shine-Dalgarno segment were modified to obtain EcoRI and BaoHI cohesive ends and this segment was inserted into plasmid pBR322 (Gene 2t 74 (1977)) after degradation with EcoRI and BamHI, whereby plasmid pGLIO1 was obtained.

35 One. plasmid such as pGLIOl or a promoter-work-Shine-

Dalgarno segment as used to construct plasmid pGL101 8202851 8 can advantageously be modified to contain the initiator codon restriction site configuration of the expression vectors of the invention. To accomplish this modification, a restriction enym site containing the initiator codon (eg ATG) as part of its recognition sequence can be added to the promoter-work-Shine-Dalgamo sequence segment. Also, a recognition sequence for an enzyme of the type may require the recognition sequence of the cleavage site by a given number. nonspecific nucleotide base pairs are used. The non-specific part of the sequence would contain the initiator codon in this case. For example, the restriction enzyme Hgal cuts at 5 and 10 base pairs. from the order GACGC *, therefore, a restriction site for this enzyme may contain the initiator codon in the nonspecific 5-10 base pair segment. Preferably, the restriction site is within three base pairs of the initiator codon, and most preferably, the restriction site is positioned so that when the restriction endonuclease is cut, the translation initiation sequence ends with the initiator codon or its complement.

The initiator codon sequence can be appropriately inserted into a plasmid or added to a linear DHA segment as a blunt-ended synthetic bridge, i.e., a segment of DHA, synthesized from individual nucleotides. For example, in the case of plasmid pGL101, this plasmid can be opened at the recognition site adjacent to the Shine-Dalgarno sequence with PvuII, after which the synthetic bridge bridge is inserted through blunt-end ligation (PvuII smooths end-splits). A preferred restriction enzyme recognition sequence is that for the enzyme Sphl. This recognition sequence is a hexanucleotide containing the ATG initiator codon. The recognition order for this enzyme is shown in Figure 8 and the cut sites are shown by a dotted line.

This new restriction site initiator codon configuration provides several distinct advantages. A unique insertion site is established in the plasmid, the presence of the initiator codon is guaranteed, and the distance between the Shine-Dalgarno sequence and the initiator codon can be precisely controlled.

Although the meaning of the distance between the Shine Dalgarno sequence and the initiator codon is not yet clear, it is believed that 8202851 "9 is particularly important for effective egg production. Preferably this distance approximates the natural distance for the protor-selling system to be used, which is typically between 0 and about 22 base pairs, preferably between 3 and 7 base pairs This distance can be controlled by the size of the synthetic bridge piece being used to control the addition of the initiator codon The sequence length can then be optimized by a selection based on the behavior of organisms containing plasmids with sequences of different lengths.

Because a gene rarely has a special restriction site at the beginning of its coding sequence, some modification of the gene is usually required before it can be incorporated into the expression vector. If the given restriction sites occur near the beginning of the gene and at or behind the end of the gene, decomposition with restriction enzyme will result in the fins of a slightly dangerous or shorter DHA segment than the actual gene. gene may be appropriately fed into the expression vector, but the protein produced by bet. organism into which the vector is inserted will differ slightly from the natural egg protein; usually, the amino acid sequence will be slightly shorter or longer than the natural egg protein. In many cases, these differences are of no significance because the resulting egg protein has substantially equal reactivity; On the other hand, when the structure of the egg protein is critical, the cut gene may preferably be modified so that its original structure is established or the resulting egg protein modified to produce the natural structure. More often, the foreign gene will not have appropriate restriction sites corresponding to the unique restriction insert site for the expression vector. In this case, the gene is cut out conventionally using one or more restriction enzymes which cut out at appropriate locations near the beginning and end of the gene. The ends of the gene are then modified by "chewing" with an exonuclease through the excess DM removed and by bridges. to. insert with base pair sequences corresponding to the restriction insertion site for the expression vector. If segments of the genes themselves have been removed in isolation of the genes, the genetic information in these segments (if known) can also be returned by means of synthetic bridges, if appropriate.

8202851 9 »10

When adding bridges with the desired restriction enzyme site for each end of the gene, each end is preferably blunted, i.e., single strand protrusions are removed. If the final modifications described above. not resulting in blunt end pieces, the end pieces may be blunted according to known procedures (Ullrich, et al.). Once a blunt-ended gene has been obtained, pre-synthesized bridges can be added by blunt-ended ligation using, for example, the enzyme DNA ligase. Subsequently, degradation of the modified gene with the restriction enzyme corresponding to the site in the linker removes any excess linker and results in a gene with cohesive ends. The modified gene can then be appropriately inserted into the transfer vector opened with the restriction enzyme. Insertion of the gene into the expression vector of the invention reforms the initiator codon.

It will be appreciated that the expression vectors described herein can take many forms. They can be used to insert a series of eucariotic genes into procariotic organisms. The production of foreign protein by this type of organism can then be placed under the control of effective promoter working systems. In addition, once a promoter-work-Shina-Dalgarno initiation codon sequence plasmid has been obtained, this entire sequence can be appropriately cloned and cut out for insertion into other vectors using known genetic techniques. For example, specialized transfer-25 vectors can be precisely adapted to specific situations.

Example I

The aim of the present experiment was. placing the gal. e (galactokinase) gene of E. eoli under the control of the lac work / promoter using an expression vector constructed according to the invention. Briefly, the operation was divided into three parts: (1) The vector pGL101 (Proc. Hatl. Acad. Sci. USA T7, 5230, 1980) was modified so that a synthetic DMA sequence encoded with the endonuclease SphI recognition site was generated. bound at a unique Pvuir site operator distal to the lac ribosome binding site, thereby obtaining a hybrid translatle initiation site with the ATG codon according to the following scheme (the new vector was attached ....... 820 2 85 1% 11 denoted as pGX951):

PvuII cleavage site pGLlOl lac o / P 'AGGAACAGGATC ____

r ---- TCCTTGTCCTAG

^ PvuII endonuclease v lac o / p AGGAACAG

----- TCCTTGTC

+ SphI "bridge- GCATGC

10 ‘piece CGTACG

Ligation

V

lac o / p AGGAACAGGCATGC ----- “TCCTTGTCCGTACG

15

SphI Restriction Ψ

Translation 20 - Initiation place i—: i

PGX951 lac o / p AGGAACAGGCATG

----- "TCCTTGTCC

(2) A restriction fragment with Ret gal K gene verd modified such that its I-terminal sequence encompassed a ligated Sphi bridge that generated a complement for a new ATG initiation codon and a second codon with all further original sequences in correction reading sequences. The following scheme illustrates the gal K gene modification: 35 8202851 12. . native N-terminal codon

Hindlll | _j AccI

plants_AGAAATGAGTCTGA. gal K gen plants

TCTTTACTCAGACT

• - i__i ·

Hinfl ^ restriction site ~ \ 5 ψ Hinfl

AGTCTGA qal K AccI

GACT

filling in DNA POLYMERASE (KLENOW)

10 restriction + dATP, dCTP, TTP

tips, f AGTCTGA.

TCAGACT

1 c + bridge- AAGCATGCTT

. - piece TTC.GTACGAA

ligation Sphl instead

V

AAGCATGCTTAGTCTGA _; __ AAGCATGCTT

2o TTCCTACGAATCAGACT TTCCTACGAA

• Sphl

V

_______ new codon., π

CTTAG.TCTGA qal K AACGATG

CTACGAATCAGACT "TTG ..

Or) (3) Ligation of (2) and (1) according to the following scheme reconstitutes a complete translation initiation site allowing an in-phase synthesis of galactokinase: 35 8202851 13

Vector: lac o / p AGGAACAGGCATG

pGX951 TCCTTGTCC

Insert 5: CTTAGTCTGA gal K. . ·

CTACGAATCAGACT

LIGATION

10 ψ

gal K.

lac o / p AGGAACAGGCATGCTTAGTC_. . .

TCCTTGTCCGTACGAATCAG

I_I

TRANSLAT IE

"5.". INITIATION

PLACE

Experiment Part 1. Preparation of the vector pGX951

Plasmid pGL101 was prepared as described by Taniguichi in Eroc. Natl. Acad. Sci. U.S.A. 77, 5230. The purified plasmid DNA was prepared from clarified cell lysates (Proc. Natl. Acad. Sci.

25 U.S.A. H, 3 ^ 55 (197M) by two successive band isolations in CsCl grafts. A reaction mixture was prepared in a volume of 50 mm and containing 15 µg of plasmid pGL101 DNA, 25 units of endonuclease PvuII (New England Biolabs) and buffer salts in the final concentrations 50 mM NaCl, 6 mM tris-HCl pH Ί6 mM MgCl ^ 10 mM mercaptoethanol and 100 µg bovine serum albumin per cm 2. This buffer is referred to below as "50 mM salt buffer". After incubation for one hour at 37 ° C, 0.8 units of calf intestine alkaline phosphatase from Boehringer, Mannheim were added and incubated for another 30 minutes at 65 ° C. The mixture was then cleaved by electrophoresis in 1% agarose (Sea-Plaque from Marine Colloids) by standard methods (J. Bacteriol. 129, 388 (1977)). After this electrophoresis, the linear plasmid was visualized by staining with ethidium bromide, from the gel is cut and extracted according to Anal Biochem.103, 26k (1980).

8202851

1U

The synthetic oligonucleotide, GCATGC (bridge) is prepared according to CGTACG J. Biol. Chem. 250, ^ 592 (1975)) · The synthetic bridge was treated with kinase in a reaction volume of 20 µl containing 5 µg bridge, 1 mM disodium ATP (PL Biochemicals), 10 units of Th polynueleotide-5 kinase (PL biochemicals), 50 mM tris-HCl with pH 7.6, 10 mM MgCl 4, 5 mM dithiothreitol, 0.1 mM spermidine (Sigma) and 0.1 mM disodium EDTA.

The reaction mixture was incubated at 37 ° C for one hour and then at 65 ° C for 5 minutes. A 1+ mm portion of this mixture was added to a mixture containing 2 µg plasmid pGL101 digested with PvuII endonuclease, 10 µmM disodium ATP, 10 mM dithiothreitol, 100 µg bovine serum albumin per cm 2. 300 units of Tk DNA ligase (Hew England Biolabs), 50 mM tris-HCl with 3 pH 7.6 and 5 mM MgClg in a volume of 20 mm. The reaction mixture was incubated at 12 ° C for 1 hour. This mixture was used to transform the E. coll ΝΊ00 strain. The cells were fitted for a transformation according to the method of Proc. Natl. Acad. Sci. U.S.A. 69, 3, 2100 (1972). A 10 mm serving of it. ligation mixture was added 3.

to 0.2 cm of an ice-cold cell suspension and incubated on ice for 1 hour.

The mixture was heat shocked at 2 ° C for 2 minutes and stored at 2 ° C for 10 minutes. Three cm 3 of the LB medium (10 g tryptone, 5 g 20 yeast extract and 10 g NaCl per liter) was added when the cells were incubated at 37 ° C for one hour. 0.1 cm 2 volumes were plated on plates with 1.5% agar, LB medium and 100 µg per cm ampicellin. After 16 hours of incubation, approximately 200 colonies were observed, compared to ten in a Control mixture without bridge. Ten of these colonies were grown overnight in 10 cm volumes of LB medium. Lysates were prepared as described. The isolated DNA was found to have been cut once by the S11 hl endonuclease but unaffected by PvuII, compared to the starting plasmid pGL101 which was not cut by SphI but once by PvuII. The new plasmid is designated as pGX951. One purified. 50 µg DNA preparation was cut by SphI and treated with calf intestine alkaline phosphatase as described above.

2. Ligation of synthetic bridge piece to a renal fragment containing bile K.

8202 85 1 ~

The source of bile E was plasmid pK0-I DNA, which had been purified as above. The plasmid and restriction data were obtained from Dr. Keith McKenney. The gst1 E gene was obtained from a restriction fragment bound by Hind III and Acc I sites (Figure 9). The reaction mixture contained "50 mM salts" buffer (see above), 50 yug pKO-I plasmid DNA and 70 units of Hindll endonuclease (Betbesda Research Labs) in a volume of 125 mm 2. After 2 hours at 37 ° C, the DM precipitated with 5 2 volumes of ethanol »recovered by centrifugation, dried and redissolved in 55 mm H« 0. A volume of 20 mm0 buffer salts (5 x concentrated "50 mM salt buffer") was added. as well as 25 mm endonuclease

AccI (10 units, Νβν England Biolabs). The incubation lasted 2.5 hours at 37 ° C and then 5 minutes at 65 ° C. The DMA was again precipitated with ethanol as above and dried. Partial decomposition then performed with endonuclase Hinfl; This was done because in addition to the specific Hinfl plants at the N-end of gal K also an inner one

Hinfl plants are contiguous, which must be left intact. The 2 ^ 0 mm reaction mixture contained 5.7 units Hinfl (Bethesda Research Lab), 9.6 µg of the Hindll-AccI fragment DNA and the "50 mM salt" buffer.

Then incubate for 2 1/2 minutes at 37 ° C followed by 15 minutes at 65 ° C. The DNA was extracted with 50 mm phenol, the residual phenol removed with ether and the DBA precipitated with 2 volumes of ethanol. The granular precipitate is volatilized with volumes of 70% ethanol and dried in vacuo. The part mixture is then filled in by DM polymer phase through the adhesive restriction ends into blunt ends then converted for ligation of the bridge. A reaction mixture of HO mm contained 3.2 µg of the Hinfii "part" DNA described above, 4 mm polymerase buffer (Bethesda Research Labs nick translation kit), 10 mM MgClg, 25 k units DNA polymerase "Klenov" ( Boehringer-Mannheim) and 0.2 mM dASP, dCTP, dGTP and TIP (Bethesda Research Labs). This mixture was evaporated at 23 ° C for 20 minutes and then incubated at 65 ° C for 15 minutes. Ligation of a synthetic oligonucleotide AAGCATGCTT1d performed after a kinase treatment

TTCGTACGAA

division of the bridge piece as described above. A reaction mixture of ^ 0:.

-5 .. mm contained "50 mM salts" buffer as above (1), 3 µg of the partially decomposed DNA, 2 µg with kinase treatment bridge and 15 units Ί & DM ligase (Nev England Biolabs) . The whole was incubated at 20 ° C for 20 hours and incubated at 65 ° C for 5 minutes. After cooling, the mixture was endowed with .20 units of SphI endonuclease and incubated for another 1 hour at 37 ° C. Then the mixture is extracted with 20 mm phenol, the remainder phenol is removed with ether and 2 volumes of ethanol are used to precipitate. the 8202851 ...............

:. - 1β DM. After washing with 70% ethanol and drying in vacuo, this DM was

O

newly dissolved in 20 mm ligase buffer (2 ud above) containing 2.5 µg of plasmid pGX951 (digested with SphI and treated with phosphatase, see above), 1000 units of DM ligase (New England Biolabs) and 0.5 mM ATP. The mixture was incubated at 20 ° C for 16 hours and at 65 ° C for 15 minutes. After cooling, 10 units of Endonuclease Narrow (Bethesda Research Labs) were added to provide an optional molecule-containing sequence between the Hindlll and Hinfl. linearization (Figure 9). After this, incubated for 1 hour at 37 ° C and 15 minutes at 65 ° C, the cooled mixture was used for a transformation of 200 mm E,. coli strain N100 gal: K which was fitted and transformed as described above. The cells were streaked on McConkey agar containing 1% galactose and 100 mg / l ampicillin. Colonies were observed after 16 hours of incubation. Red colonies now capable of expressing the gal K gene were purified and made as DNA preparations over 15. This DNA was subjected to a restriction analysis to check the expected pattern. The restriction map of plasmid pK0-1 in the gal E region is shown in Figure 9 of the drawing. The figures in Figure 1 refer to the distance in base pairs. Symbols: E = endonuclease AcoRI, H = Hindlll, HF = Hinf I, A * Accl, 20 S = Narrow. The line marked "gal K" indicates the location of the gal K (galactokinase) gene. The galactokinase producing strain thus obtained is designated pGx951 and deposited with the ATCC under number 31917 ~ 020 2 851 '

Claims (22)

1 »Expression vector for inserting a gene. in a procariotic organism comprising a closed loop DHA with signals that allow it to multiply in a procariotic host cell and further: (a) promoter, working and translation initiation sequences recognizable by the procariotic host cell; (b) a Shine-Dalgarno sequence and an initiator codon within the translation initiation sequence; and (c) a recognition sequence for a restriction endo-nuclease that cleaves near the initiator codon by preserving this initiator codon or its complement within the translation initiation sequence. Expression vector according to claim 1, characterized in that the restriction endonuclease is within three base pairs of the. initiator codon or cuts its complement. Expression vector according to claim 1, characterized in. that the restriction endonuclease cuts open such that the translation initiation sequence ends with the initiator codon or its complement. U. Expression vector according to claim 1, characterized in that the initiator codon is present within the recognition sequence for the restriction endonuclease. Expression vector according to claim 1, characterized in that the initiator codon is present in the recognition order for the restriction endonuclease Sphl. Expression vector according to claims 1 to 3, characterized in that the restriction endonuclease recognition sequence is separated from the restriction endonuclease cleavage site by a number of nonspecific nucleotide base pairs. Expression vector according to claim 6, characterized in that the restriction is endonuclease ffgal. Expression vector according to claims 1 to 5, characterized in that the initiator codon of the Shine-Dalgarno sequence is separated by 0 to about 22 base pairs. Expression vector according to claim 8, characterized in that ..... ¢ 202851 ----------------- - that an initiator codon of the Shine-Dalgarno sequence cleaved. is by 3 - T base pairs. Expression vector according to claim 1, characterized in that the gene is a procariotic gene, a eucariotic gene or a synthetic DM sequence encoding a specific polypeptide. Expression vector according to claims 1 to 5, characterized in that the procariotic host cell is Escherichia coli and the promoter, promoter and Shine-Dalgarno sequence is derived from the lac or trp operons of E. coli. Expression vector according to claims 1 to 5, characterized in that the. procariotic host cell is Escherichia coli and the promoter, promoter, and Shine-Dalgarno sequences are derived from a hacteriophage. 13. Bacterium of the genus Escherichia containing an expression vector according to claim 11. 11. Bacteria of the genus Escherichia containing an expression vector according to claim 12. • Almost pure DM segment consisting of: (a) promoter, sales and translation initiator sequences recognizable by a procariotic host organism; (b) a Shine-Dalgarno sequence and an initiator codon within the translation initiation sequence and (c) a restriction endo-nuclease recognition sequence that cleaves close to the initiator codon or its complement within the translation initiation order. DM segment according to claim 15, characterized in that the restriction endonuclese cuts open within three base pairs of the initiator codon or its complement. DM segment according to claim 15, characterized in that the restriction endonuclease cuts open such that the translation initiation sequence ends with the initiator codon or its complement. DM segment according to claim 15, characterized in that the initiator codon is present within the restriction endonuclease recognition sequence. 19. DM segment according to claim 15, characterized in that the initiator codon is present in the recognition sequence for the restricon-endonuclease Sphl. 20. DNA segments according to claims 15, 16 or 17, characterized in that the restriction endonuclease recognition sequence is separated from the restriction endonuclease cleavage site by a number of nonspecific nucleotide base pairs. DNA segment according to claim 20, characterized in that the restriction endonuclease is Hgal. DNA segment according to claims 15-19, characterized in that the initiator codon is separated from the Shine-Dalgarno sequence 10 by 0 to about 22 "base pairs. DNA segment according to claim 22, characterized in that the initiator codon of the Shine-Dalgarno sequence is separated by 3-7 base pairs. 2k. DNA segment according to claims 15-19, characterized in that the promoter, sales and Shine-Dalgarno sequences are derived from the lac or trp operons of E. coli. DNA segment according to claims 15-19, characterized in that the promoter, sales and Shine-Dalgamo sequences are derived from a bacteriophage. 82 0 2 8 5 1 .......