KR20090039988A - Production of recombinant human hemoglobin using pichia yeast - Google Patents

Abstract

A production method of recombinant human hemoglobin using pichia yeast is provided to be safe in the human body without mixing of unknown virus and to massively produce human hemoglobin by using pichia yeast. A production method of recombinant human hemoglobin using pichia yeast comprises the steps of (i) transforming pichia yeast with an expression vector containing DNA coding human hemoglobin alpha chain and an expression vector containing DNA coding human hemoglobin beta chain, wherein the expression vectors are in the control of a promoter functioning in pichia yeast; (ii) culturing the transformed pichia yeast in the culture medium; and (iii) collecting the human hemoglobin from the obtained culture material. The DNA coding the human hemoglobin alpha chain and the DNA coding the human hemoglobin beta chain are exist in the same vector. The pichia yeast is Pichia pastoris.

Description

Production of Recombinant Human Hemoglobin Using Pichia Yeasts {PRODUCTION OF RECOMBINANT HUMAN HEMOGLOBIN USING PICHIA YEAST}

The present invention relates to the production of recombinant human hemoglobin and expression vectors therefor. More specifically, the present invention provides a method for producing human hemoglobin using Pichia yeast, and an expression cassette comprising DNA encoding human hemoglobin β chain and an expression cassette comprising DNA encoding human hemoglobin α chain. It relates to an expression vector containing (to be aligned back and forth).

Hemoglobin is a protein that contains a high concentration of red blood cells, and has the function of transporting various gas molecules including oxygen. Structurally, it is a heterotetramer which consists of two alpha chains and two beta chains.

Red blood cell replacement is being developed as a preparation using hemoglobin. Until now, hemoglobin vesicles, which are intramolecular cross-linked, polymerized, polymer-bonded, or hemoglobin-encapsulated hemoglobin, have been devised. In particular, hemoglobin endoplasmic reticulum has been aimed at practical use as a lifesaving oxygen solution by single administration from biological data such as shape, hemoglobin purity, physical data such as safety, retention in the body, dynamics of the body and oxygen transport ability.

On the other hand, preparations using hemoglobin derived from human blood may cause the incorporation of unknown viruses and the like, and there is a problem in terms of their use to the human body in terms of safety. However, there is a possibility that hemoglobin preparations can be provided safely by microorganisms transformed using genetic recombination techniques.

Until now, in the production of genetically modified hemoglobin, a system using Escherichia coli as a host cell was mainly used. However, in this technique, if (1) an enzyme such as methionine aminopeptidase or the like is not co-expressed with hemoglobin, more first methionine is added to the N-terminal than in the natural form, (2) There has been a problem that aminolevulinic acid must be added to the culture solution for the purpose of heme synthesis.

Two examples have been reported so far for the production of recombinant hemoglobin using Saccharomyces cerevisiae (see Non-Patent Documents 1 and 2). In this system, since the first methionine is cleaved by an enzyme possessed by S. cerevisiae itself, the native form and the primary sequence are completely identical, and it is not necessary to add aminolevulinic acid for heme synthesis. However, this method also cannot be said to be sufficient due to problems in yield and the like, and it has not yet reached industrial production of recombinant human hemoglobin.

Pichia yeast is known as one of the industrial yeasts other than Saccharomyces genus. This yeast is capable of propagating with methanol as the sole carbon source, and when grown in methanol, enzymes necessary for the treatment of methanol and its metabolic intermediates are de-inhibited and expressed. Production of heterologous proteins using this methanol magnetization pathway has been studied and has already been applied to the production of albumin for blood preparations (see, for example, Japanese Patent Laid-Open No. 6-22784). It is known that the expression amount is very high and about 10 g of albumin can be manufactured in 1 L of medium.

However, there are often cases where the expression efficiency of the heterologous protein is low due to the combination (homogeneity) of the target heterologous protein and the host cell. In particular, in the case of heterooligomeric proteins such as hemoglobin, many conditions such as the balance of the amount of expression of each subunit and oligomer formation must be made clear, and it is not easy to obtain desired high expression. Indeed, there are very few examples of Pichia yeast being used for the industrial production of human proteins.

<Non Patent Literature 1>

Wagenbach, M. et al., Bio / Technol., 9: 57-61 (1991)

<Non Patent Literature 2>

Coghlan, D. et al., Eur. J. Biochem., 207: 931-936 (1992)

An object of the present invention is to provide a means for easily and in large quantities producing recombinant proteins such as safe human hemoglobin, albumin, globulin, etc., which have sufficient functions and activities as in the natural form and are free of virus incorporation.

As a result of intensive studies to achieve the above object, the present inventors have found that DNAs encoding a plurality of proteins or peptide chains, such as expression vectors containing DNAs encoding α and β chains of human hemoglobin, are contained. As an expression vector, Pichia pastoris , one of the genes of the genus Pichia , is transformed, and the obtained transformant is cultured in a medium, so that various proteins or peptides such as human hemoglobin, especially heterooligomeric proteins, etc., are produced. It succeeded in collect | recovering and came to complete this invention.

That is, the present invention,

(1) an expression vector containing DNA encoding a human hemoglobin α chain under the control of a promoter capable of functioning in Pichia yeast, and a human hemoglobin β chain under the control of a promoter capable of functioning in Pichia yeast A method for producing a functional recombinant human hemoglobin, comprising culturing a Pichia yeast transformed with an expression vector containing a DNA to be cultured in a medium and recovering human hemoglobin from the obtained culture;

(2) the method according to the above (1), wherein the DNA encoding the human hemoglobin α chain and the DNA encoding the human hemoglobin β chain are on the same vector;

(3) the method according to the above (2), wherein the DNA encoding the human hemoglobin β chain is located upstream in the transcription direction;

(4) the method according to any one of (1) to (3) above, wherein the DNA encoding the human hemoglobin α chain and the DNA encoding the human hemoglobin β chain are under the control of a separate promoter;

(5) The method according to any one of (1) to (4) above, wherein the DNA encoding the human hemoglobin α chain and the DNA encoding the human hemoglobin β chain are under the control of the alcohol oxidase 1 promoter;

(6) the method according to the above (5), wherein the transformed Pichia yeast is cultured in a liquid medium containing methanol as a single carbon source;

(7) The method according to the above (6), wherein the culture is a fed-batch culture;

(8) The method according to any one of (1) to (7), wherein the Pichia yeast is Pichia pastoris; And

(9) From the upstream side of the transcription direction, DNA encoding the alcohol oxidase 1 promoter, DNA encoding human hemoglobin β chain, terminator capable of functioning in Pichia yeast, alcohol oxidase 1 promoter, DNA encoding human hemoglobin α chain, and Expression vectors containing terminators that can function in Pichia yeast

To provide.

The present invention also provides

(10) A method for producing an expression vector containing DNA encoding a plurality of proteins under the control of a promoter capable of functioning in Pichia yeast, wherein when ligating the vector and the DNA encoding one of the plurality of proteins, A ligation reaction is performed without dephosphorylation of the vector;

(I) an expression vector containing DNA encoding at least one protein under the control of a promoter capable of functioning in Pichia yeast, wherein said expression vector is cleaved at one place other than inside said promoter and said DNA; As a DNA encoding another protein under the control of a promoter capable of functioning in Pichia yeast, which provides an expression vector that is digested by a restriction enzyme which is capable of functioning and whose resulting both ends are not dephosphorylated. Wherein both ends provide DNA complementary to the ends of the expression vector of (i), and (iii) the ligation reaction between the expression vector of (i) and the DNA of (ii) is performed. The method described in 10);

(12) A recombinant protein comprising recovering the plurality of proteins from a culture obtained by culturing Pichia yeast transformed with the expression vector obtained by the method described in the above (10) or (11) in a medium. Method of preparation;

(13) The method according to the above (12), wherein the plurality of proteins are polymerized in a culture to form a functional protein complex.

And so on.

Since the human hemoglobin provided by the method of the present invention is produced by genetic recombinant technology using Pichia yeast as a host, there is no fear of incorporation of unknown virus, which is a problem peculiar to blood-derived products, and can be used safely in the human body. . In addition, by using Pichia yeast, a large amount of human hemoglobin can be produced and have the same function and properties as the natural type.

As the DNA encoding the human hemoglobin α chain (hereinafter sometimes abbreviated as “Hbα”) used in the present invention, a nucleotide sequence encoding an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is used. DNA encoding the human hemoglobin β chain (hereinafter sometimes abbreviated as “Hbβ”) is a DNA containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 4. And DNAs each containing a nucleotide sequence.

As an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 (or SEQ ID NO: 4), at least about 80%, preferably about 90% or more of the amino acid sequence represented by SEQ ID NO: 2 (or SEQ ID NO: 4), More preferably about 95% or more, particularly preferably about 97% or more, amino acid sequences and the like. As used herein, "homology" refers to an optimal alignment in which two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is applied to one or both sides of the sequence for optimal alignment). The ratio of the same amino acid residue to the total amino acid residues overlapping with respect to the overlapping total amino acid residues (%) in the gap). "Amino acid" means similar amino acids in physical and chemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids Amino acids classified into the same group (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids having a hydroxyl group (Ser, Thr), and small side chain amino acids (Gly, Ala, Ser, Thr, Met) Can be mentioned. Substitution by such analogous amino acids is expected to result in no change in the phenotype of the protein (ie, conservative amino acid substitutions). Specific examples of conservative amino acid substitutions are well known in the art and described in various documents (see, eg, Bowie et al., Science, 247: 1306-1310 (1990)).

Homology of the amino acid sequence in the present specification, using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool), the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; Filtering = OFF). Other algorithms for determining homology of amino acid sequences include, for example, Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993), which is included in the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997)), Needleman et al., J. Mol. Algorithm described in Biol., 48: 444-453 (1970), which is included in the GAP program in the GCG software package, and in Myers and Miller, CABIOS, 4: 11-17 (1988). Algorithms are included in the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package], Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988), which algorithm is included in the FASTA program in the GCG software package, and the like, and these may likewise be preferably used.

More preferably, the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 (or SEQ ID NO: 4) is about 80% or more, preferably at least about the amino acid sequence represented by SEQ ID NO: 2 (or SEQ ID NO: 4). At least about 90%, more preferably at least about 95%, particularly preferably at least about 97%.

A protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 (or SEQ ID NO: 4) includes an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 (or SEQ ID NO: 4) described above. And a protein having substantially the same activity as the protein comprising the amino acid sequence represented by SEQ ID NO: 2 (or SEQ ID NO: 4).

Substantially homogeneous activity includes the physiological activity of human hemoglobin, for example, the activity of carrying gas molecules such as oxygen, and the like. "Substantially homogeneous" means that these activities are qualitatively the same. Therefore, it is preferable that the activities such as the oxygen transporting ability are equivalent (for example, about 0.5 to about 2 times), but the quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.

The measurement of the activity (for example, automatic oxidation rate constant, oxygen affinity, cooperativeness, etc.) such as oxygen transport ability can be performed according to a method known per se, for example, the method described in Japanese Patent Publication No. 2004-500871, but is not limited thereto.

In addition, Hbα (or Hbβ) used in the present invention includes, for example, (1) one or two or more (preferably about 1 to 30 or more) amino acid sequences represented by SEQ ID NO: 2 (or SEQ ID NO: 4). Preferably an amino acid sequence of about 1 to 10, more preferably 1 to several (2, 3, 4 or 5) amino acids deleted, (2) represented by SEQ ID NO: 2 (or SEQ ID NO: 4) One or two or more amino acids (preferably between about 1 and 30, more preferably between 1 and 10, and even more preferably between 1 and 2 (2, 3, 4 or 5)) in the amino acid sequence 1 or 2 or more (preferably about 1 to 30, more preferably about 1 to 10, and even more) the amino acid sequence represented by this added amino acid sequence and (3) SEQ ID NO: 2 (or SEQ ID NO: 4). More preferably, amino acid sequence in which 1 to 2 (2, 3, 4 or 5) amino acids are inserted, (4) SEQ ID NO: 2 ( Is one or two or more (preferably about 1 to 30, more preferably about 1 to 10, and still more preferably 1 to 2 (2, 3, 4) of the amino acid sequence represented by SEQ. Or a protein containing an amino acid sequence in which 5) amino acids are substituted with another amino acid, or (5) an amino acid sequence combining these amino acids.

When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the activity of the protein is maintained.

Human hemoglobin in the present invention is used not only in adult hemoglobin (HbA1), but also in the sense of including HbA2 in which the α chain and the δ chain are combined, and fetal hemoglobin (HbF) in which the α and γ chains are combined. Therefore, in the production of HbA2 or HbF, DNA encoding the δ or γ chain is used instead of the β chain. The amino acid sequence and base sequence of the δ chain are registered, for example, in GenBank Accession Nos. NP_000510 and NM_000519. On the other hand, the amino acid sequence and base sequence of the γ chain are registered, for example, under GenBank accession numbers NP_000550 and NM_000559.

Examples of the DNA encoding Hbα (or Hbβ) include human genomic DNA, adult cDNA derived from Hbα (and / or Hbβ) producing cells (eg, red blood cells, etc.), synthetic DNA, and the like. Genomic DNA and cDNA encoding Hbα (or Hbβ) are used as genomic DNA fractions prepared from their producing cells or tissues (e.g., blood) and total RNA or mRNA fractions as templates, respectively, to polymerase chain reaction. (Hereinafter, abbreviated as "PCR method") and reverse transcriptase-PCR (hereinafter, abbreviated as "RT-PCR method"). Alternatively, genomic DNA and cDNA encoding Hbα (or Hbβ) can be colonized or plaqued from genomic DNA libraries and cDNA libraries prepared by inserting genomic DNA prepared from the cells and tissues described above and fragments of whole RNA or mRNA into appropriate vectors. It can also be cloned, respectively, by a hybridization method, PCR method, etc. The vector used for the library may be any of bacteriophage, plasmid, cosmid, and phagemid.

As the DNA encoding Hbα (or Hbβ), for example, DNA containing the nucleotide sequence represented by SEQ ID NO: 1 (or SEQ ID NO: 3), or stringent with the nucleotide sequence represented by SEQ ID NO: 1 (or SEQ ID NO: 3) And DNA encoding a protein containing a base sequence that hybridizes under the conditions and having substantially the same activity (for example, oxygen transport ability) as the above-described Hbα (or Hbβ).

As DNA that can hybridize under stringent conditions with the nucleotide sequence represented by SEQ ID NO: 1 (or SEQ ID NO: 3), for example, about 70% or more of the nucleotide sequence represented by SEQ ID NO: 1 (or SEQ ID NO: 3), preferably DNA containing a base sequence having at least about 80%, more preferably at least about 90%, particularly preferably at least about 95% homology, and the like are used.

Homology of the base sequence in the present specification, using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool), the following conditions (expected value = 10; allow gap; filtering = ON; Match score = 1; mismatch score = -3). As another algorithm for determining the homology of the nucleotide sequence, the above-described homology calculation algorithm of the amino acid sequence can be preferably exemplified.

Hybridization can be carried out according to a method known per se or a method equivalent thereto, such as the method described in Molecular Cloning Second Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). In addition, when using a commercially available library, hybridization can be performed in accordance with the method as described in the attached instruction manual. Hybridization can be performed preferably under stringent conditions.

As highly stringent conditions, the hybridization reaction at 45 degreeC in 6xSSC (sodium chloride / sodium citrate), for example, 1 time of washing at 65 degreeC in 0.2xSSC / 0.1% SDS, etc. are mentioned. Those skilled in the art can appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the cleaning solution, the temperature of the cleaning, and the like, It can be easily adjusted to the desired stringency.

DNA encoding Hbα (or Hbβ) is amplified by PCR using synthetic DNA primers having a portion of the nucleotide sequence encoding Hbα (or Hbβ), or DNA inserted into a suitable expression vector is expressed by Hbα ( Alternatively, the DNA fragment can be cloned by hybridizing a DNA fragment or a synthetic DNA encoding a part or entire region of Hbβ).

The DNA encoding Hbα (or Hbβ) can be obtained from human genomic DNA or RNA (cDNA) as described above, and chemically synthesizes DNA strands or partially overlaps oligo DNA single strands synthesized by PCR. By connecting using, it is also possible to construct DNA encoding the full length of Hbα (or Hbβ). The advantage of constructing full-length DNA in combination with chemical synthesis or PCR is that the codon can be designed over the full length of the gene for the host into which the gene is introduced. A plurality of codons encoding the same amino acid are not used uniformly and their frequency of use varies depending on the species. In general, codons contained in genes that are highly expressed in a certain species contain a lot of codons with a high frequency of use, whereas genes with low expression levels are a bottleneck due to the presence of a low frequency of use. There are many examples that hinder expression. In expressing heterologous genes, there have been many reports of increased heterologous protein expression levels by replacing the gene sequence with codons frequently used in host organisms. It is expected to be effective in increasing the

For the above reasons, DNA encoding Hbα (or Hbβ) can be transformed into suitable codons (ie, high frequency of use in the host) by the Pichia yeast into which it is introduced. The codon usage of each Pichia yeast is defined as the rate at which each codon is used in the total genes present on the host's genomic sequence, for example expressed as the number of uses per 1000 codons. The codon usage frequency can also be calculated approximately from the sequence of a plurality of representative genes in an organism in which the entire genome sequence is not elucidated. Codon usage data in Pichia yeast to be recombined is, for example, a genetic code usage frequency database (http://www.kazusa.or.jp/codon/) published on the homepage of the Kazusa DNA Research Institute. index.html) may be used, or a document describing the codon usage frequency in each Pichia yeast may be referred to, or the codon usage data of the Pichia yeast to be used may be determined by itself. With reference to the obtained data and the gene sequence to be introduced, the codons used in the gene sequence may be replaced with a codon having a high frequency of use for the same amino acid.

As a further advantage of constructing DNA encoding the full length of Hbα (or Hbβ) by chemically synthesizing the DNA chain or connecting the partially overlapping oligo DNA single chain by PCR method, the subsequent expression vector This makes it easy to build. For example, in the examples described below, the expression cassettes of Hbβ and the expression cassettes of Hbα are tangentially connected by using the complementarity of the adhesion ends generated by restriction enzymes BamH I and Bgl II digestion (see FIG. 1). Using complementarity of the sticky ends, multiple copies of the expression cassette can be repeatedly inserted into the vector in tandem. Therefore, the DNA encoding Hbα and the DNA encoding Hbβ preferably do not include a BamH I recognition site. For example, Hbβ cDNA registered as GenBank accession number NM_000518 in the NCBI database is located at positions 347-352. Has a BamH I recognition site (GGATCC). On the other hand, in the nucleotide sequence shown in SEQ ID NO: 3, the nucleotide sequence of the corresponding moiety (base numbers 297 to 302) is TGACCC, and a mutation is introduced so as not to include the BamH I recognition site. In addition, in the Examples described later, in order to construct an expression cassette by inserting DNA encoding Hbα and DNA encoding Hbβ between a promoter and a terminator that can function in Pichia yeast, an EcoR I recognition sequence is used to construct an Hbα. The DNA to be encoded and Hbβ are added to both ends of the DNA to be encoded (see FIG. 2). Therefore, the DNA encoding Hbα and the DNA encoding Hbβ preferably do not include an EcoR I recognition site. However, for example, Hbβ cDNA registered as GenBank accession number NM_000518 in the NCBI database is located at positions 414 to 419. In EcoR I recognition site (GAATTC). On the other hand, in the nucleotide sequence shown in SEQ ID NO: 3, the nucleotide sequence of the corresponding moiety (base numbers 370 to 375) is GAGTTT, and a mutation is introduced so as not to include the EcoR I recognition site. When modifying the restriction enzyme recognition site in the protein code sequence in this manner, it is preferable to replace the base so as not to change the amino acid sequence of Hbα (or Hbβ) by using the codon weight.

DNA cloned as described above can be used as it is, or as desired, after digestion with a restriction enzyme or after addition of a linker. The DNA may have ATG as a translation start codon on its 5 'end side and TAA, TGA or TAG as a translation end codon on its 3' end side. These translation start codons and translation end codons can be added using a suitable synthetic DNA adapter.

An expression vector containing DNA encoding Hbα (or Hbβ) is a promoter in an appropriate expression vector using, for example, a DNA fragment encoding Hbα (or Hbβ) cloned as described above using restriction enzymes and DNA ligase. It can be produced by connecting downstream.

The vector to be used is not particularly limited as long as it is genetically stable by autonomously copying or inserting into the yeast genome of bacteria of the genus Pichia yeast. As a vector which can be autonomously replicated, an YEp vector, a YRp vector, a YCp vector, etc. are mentioned, for example. Moreover, as a vector which can be inserted in a yeast genome, a YIp vector and a YRp vector are mentioned, for example.

Examples of promoters capable of functioning in Pichia yeast include promoters derived from yeast, such as PHO5 promoters derived from S. cerevisiae, PGK promoters, GAP promoters, and ADH promoters, such as alcohol oxidases derived from P. pastoris (AOX). 1 promoter, AOX2 promoter, dihydroxyacetone synthase promoter, P40 promoter, ADH promoter, folate dehydrogenase promoter, etc. are mentioned. In addition, mutant promoters, such as mutant AOX2 (mAOX2) promoters, wherein the yeast derived promoter is modified to further improve gene expression efficiency [Ohi et al., Mol. Gen. Genet., 243, 489-499 (1994); Japanese Patent Laid-Open No. 4-299984]. Preferably, the promoter is more preferably an AOX1 promoter, such as promoters of enzyme genes required for the treatment of methanol or its metabolic intermediates, such as the AOX1 promoter and the mAOX2 promoter, to utilize the methanol metabolic system of the Pichia yeast.

Expression vectors containing the DNA encoding the Hbα (or Hbβ) of the present invention can also be used for the selection of transcription termination sequences (terminators) (eg, AOX1 terminators, etc.), enhancer sequences, and yeast that can function in Pichia yeast. Selectable marker genes (nutrition requirement genes such as HIS4, LEU2, ARG4, URA3 genes from P. pastoris or S. cerevisiae, or antibiotic resistance genes such as cycloheximide, G-418, Genes resistant to chloramphenicol, bleomycin, hygromycin and the like), and the like, and may contain a replicable unit capable of functioning in yeast if desired. In addition, for mass preparation of the vector, it is more preferable to include a selection marker gene (eg, a gene for resistance to ampicillin or tetracycline, etc.) that can be used to select a replicable unit capable of functioning in E. coli and E. coli. Do.

When the expression vector is a vector of the type inserted into the yeast genome, the vector preferably further comprises a sequence homologous to the yeast genome necessary for homologous recombination. As such a homologous sequence, the sequence of the above-mentioned nutritive gene is mentioned. Therefore, in one preferred embodiment, the expression vector of the present invention refers to the expression cassette of the Hbα (or Hbβ) in the nutritive gene (herein, "expression cassette" means a unit that enables gene expression, and a promoter The protein code sequence is placed under the control of a minimum unit, but preferably a unit consisting of a promoter-protein code region-terminator is inserted (see FIG. 3).

The expression cassette of Hbα and the expression cassette of Hbβ may be supported on separate vectors or may be supported on the same vector. In the former case, the two vectors preferably contain different selection marker genes. However, in a preferred embodiment of the present invention, the expression cassette of Hbα and the expression cassette of Hbβ are arranged on the same vector. In this case, the expression cassette of Hbα and the expression cassette of Hbβ may be arranged in any order. For example, although it may be arranged head-to-head or tail-to-tail on the reverse chain, it is preferably arranged in the same transcription direction, and more preferably from the upstream side of the transcription direction, in the order of the expression cassette of Hbβ and the expression cassette of Hbα. Is placed. When DNA encoding Hbα and DNA encoding Hbβ are transcribed in the same direction, the transcription may be made from monocistronic from the same promoter or from diistronic from separate promoters. You may. When transcribed to monocistronic, the expression vector does not include a terminator on the 3 'side of the upstream code sequence. It is also desirable to contain an internal ribosome entry site (IRES) on the 5 'side of the downstream code sequence. However, in a preferred embodiment of the present invention, transcription of the DNA encoding Hbα and the DNA encoding Hbβ consists of disistrones from separate promoters.

In a particularly preferred embodiment of the present invention, from the upstream side of the transcription direction, the AOX1 promoter, the DNA encoding Hbβ, the terminator (preferably AOX1 terminator) capable of functioning in Pichia yeast, the AOX1 promoter, the DNA encoding Hbα And terminators (preferably AOX1 terminators) which can function in Pichia yeast are used.

When constructing an expression vector in which the expression cassette of Hbβ and the expression cassette of Hbα are connected in tandem, it is usually immediately upstream of the promoter of the expression vector including one expression cassette (when the expression cassette of Hbα is included) or Immediately downstream of the terminator (if containing an expression cassette of Hbβ) was linearized by cleavage with one appropriate restriction enzyme (e.g., Hbβ-pAO815 was cleaved with BamH I in FIG. 2) and the same sticky ends The other expression cassette (e.g., in FIG. 2, the Hbα expression cassette is cut by Hamα-pAO815 from BamH I and Bgl II double digestion) and DNA ligase. Ligation using agent. At this time, in general, dephosphorylation treatment (eg, calf small intestinal alkali phosphatase (CIAP) treatment) at the end is performed to prevent self-ligation on the vector side, but in order to increase the overall ligation efficiency, the dephosphorylation treatment is not performed. There is a rather preferable case.

The expression vectors obtained as described above are, for example, the competent cell method, the protoplasm method, the calcium phosphate coprecipitation method, the polyethylene glycol method, the lithium method, the electroporation method, the microinjection method, the liposome fusion method, and the particle total method (particle). known transformation techniques can be introduced into target Pichia yeast cells.

Pichia yeast to be used in the present invention is not particularly limited, for example, P. pastoris, Pichia acaciae , Pichia angusta , Pichia anomala, Pichia capsuleata ( Pichia capsulata ), Pichia ciferrii , Pichia etchellsii , Pichia fabianii , Pichia farinosa , Pichia guilliermondii ), Pichia inositovora , Pichia jadinii , Pichia methanolica , Pichia norvegensis , Pichia ofunaensis , Pichia pinus etc. are mentioned. Preferably, P. pastoris Pas, particularly auxotrophic mutant P. pastoris Pas state (e.g., P. pastoris GTS115 Pas state ^{(HIS4 -) [NNRL Y-} 15851], P. pastoris GS190 Pas state (ARG4 ^-) [NNRLY-18014), P. Paz pastoris PPF1 (HIS4 ^- a) such ^{NNRL Y-18017]) -,} URA4.

Transformed Pichia yeast can be produced by functional human hemoglobin by culturing in a method commonly used in the art. The medium to be used needs to contain at least a carbon source and an inorganic or organic nitrogen source necessary for the growth of the host cell. Examples of the carbon source include methanol, glycerol, glucose, sucrose, dextran, soluble starch, and the like. In addition, examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn leachate, peptone, casein, meat extract, yeast extract, soybean gingiva, varicose extract and the like. If desired, other nutrients such as calcium chloride, sodium dihydrogen phosphate and magnesium chloride may be contained, vitamins such as biotin, antibiotics and the like.

As a medium used, a normal natural medium (for example, YPD medium, YPM medium, YPG medium, etc.) or a synthetic medium is mentioned, for example. The pH and culture temperature of the medium may be suitably employed as the pH and temperature appropriate for the growth of yeast and the production of human hemoglobin, for example, pH of about 5 to about 8, culture temperature of about 20 ℃ to about 30 ℃. have. In addition, if necessary, aeration or agitation may be performed. Incubation usually takes about 48 hours to about 120 hours.

For example, in the case of using a promoter induced by methanol, such as an AOX1 promoter, a mAOX2 promoter, or the like as a promoter capable of functioning in Pichia yeast cells, it contains glycerol as a carbon source for the growth of the cells, and the expression of Hbα and Hbβ. Most preferably exemplified is a method of liquid aeration stirred cultivation using a natural medium controlled to pH about 6.0, containing methanol as an inducer. If the expression of Hbα and Hbβ is not desirable for the growth of the cells, a culture method of first inducing the expression of Hbα and Hbβ by increasing the amount of the cells to a carbon source other than methanol and then adding methanol is more preferable. In addition, in the culture in a jar fermentor, a high density culture method can be exemplified as a method suitable for the production of human hemoglobin. The cultivation may be carried out by any of batch culture, fed-batch culture, and continuous culture, but preferably, a fed-batch culture method may be mentioned. That is, for a certain period of time, the host cells are cultured in a medium (initial medium) containing a carbon energy source (e.g., glucose, etc.) and / or a nutrient source suitable for the growth thereof, and depending on the situation, at some point in time, the expression of Hbα and Hbβ dominates. A method in which the human hemoglobin is not taken out of the system until the end of the cultivation while further supplying a substrate (ie, methanol) to the medium is used (see, for example, Japanese Patent Laid-Open No. 3-83595).

Human hemoglobin produced in the culture can be recovered by centrifugation and / or filtration of the culture after the completion of culture to recover the yeast cells, and then isolated and purified from the cells according to a method known per se. As such a method, the method of using solubility, such as salting out and a solvent precipitation method; A method using mainly molecular weight differences such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis; A method of using a charge difference such as ion exchange chromatography; Methods using specific affinity such as affinity chromatography; A method of using a hydrophobic difference such as reverse phase high performance liquid chromatography; The method of using the difference of isoelectric points, such as an isoelectric point electrophoresis method, etc. is used. These methods may be combined as appropriate.

As a confirmation method of isolated and purified human hemoglobin, a well-known western blotting method, an activity measurement method, etc. are mentioned. In addition, purified hemoglobin can clarify its structure by amino acid analysis, N-terminal amino acid sequence, primary structure analysis, and the like. Human hemoglobin thus obtained has a primary structure (i.e., cleavage removal of the first methionine) and a higher order structure like the natural type, and also exhibits the same physiological activity as the natural type.

The recombinant human hemoglobin prepared in the present invention can be administered to a living body, for example, as a red blood cell replacement. When administered as an erythrocyte substitute, hemoglobin may be administered by encapsulating it in liposomes or by dispersing it in an emulsion. As for the technique of such formulation and the method of administration to humans, for example, Japanese Patent Laid-Open No. 2004-307404, Japanese Patent Laid-Open No. 2006-104069, Japanese Patent Laid-Open No. 2002-161047, Japanese Patent Laid-Open No. 2001-348341, and Japanese Patent Laid-Open It describes in 08-003063, Unexamined-Japanese-Patent No. 08-003062, Unexamined-Japanese-Patent No. 07-017874, Unexamined-Japanese-Patent No. 06-321802, Unexamined-Japanese-Patent No. 06-072892, etc.

Sequence Table Free Text

SEQ ID NO: 5

primer.

SEQ ID NO: 6

primer.

SEQ ID NO: 7

primer.

SEQ ID NO: 8

primer.

SEQ ID NO: 9

primer.

SEQ ID NO: 10

primer.

SEQ ID NO: 11

primer.

SEQ ID NO: 12

primer.

SEQ ID NO: 13

Hbα cDNA having EcoR I sites at the 5 'and 3' ends.

SEQ ID NO: 14

Hbβ cDNA with EcoR I sites at the 5 'and 3' ends.

SEQ ID NO: 15

primer.

SEQ ID NO: 16

primer.

SEQ ID NO: 17

primer.

SEQ ID NO: 18

primer.

SEQ ID NO: 19

primer.

Although an Example is given to the following and this invention is demonstrated to it further more concretely, these are simple illustrations and do not limit the scope of this invention at all.

Example

(1) amplification of the hemoglobin gene

A plasmid in which a human hemoglobin gene has been introduced into the plasmid pBEX (hereinafter referred to as "pBEX-Hb") can be used as a template, and each of the hemoglobin α chains is used as a template. Hbα-sense primer of SEQ ID NO: 5 and Hbα-antisense primer of SEQ ID NO: 6 for the DNA sequence, and Hbβ-sense primer of SEQ ID NO: 7 and Hbβ-antisense primer of SEQ ID NO: 8 for the DNA sequence of the hemoglobin β chain The primer was used as a mutation of the EcoR I restriction enzyme site (QuikChange XL Site-Directed Mutagenesis Kit, Stratagene), which interfered in subsequent operations. As the reaction conditions for the mutation, the DNA was treated at 95 ° C. for 30 seconds, followed by 12 cycles of denaturation (95 ° C., 30 seconds), annealing (55 ° C., 1 minute), and extension (68 ° C., 10 minutes). It was. After the reaction, the plasmid of the template was digested by Dpn I, and the resulting pBEX-Hb (αΔ) and pBEX-Hb (βΔ) were introduced into XL-10-gold ultracompetent cells and transformed. The transformants into which the target plasmids pBEX-Hb (αΔ) and pBEX-Hb (βΔ) were introduced were screened in ampicillin-added medium, and the plasmids were purified from the resulting transformants (QIAprep Spin Miniprep Kit, manufactured by QIAGEN). The plasmid was digested with EcoR I (manufactured by Takarashuzo) to confirm that mutations were occurring.

Using the obtained pBEX-Hb (αΔ) and pBEX-Hb (βΔ) as templates, the Hbα-sense primer of SEQ ID NO: 9, the Hbα-antisense primer of SEQ ID NO: 10, and the hemoglobin β chain, respectively, to the DNA sequence of the hemoglobin α chain The DNA sequence of was subjected to PCR using polymerase (KOD-plus-, Toyoboseki) using the Hbβ-sense primer of SEQ ID NO: 11 and the Hbβ-antisense primer of SEQ ID NO: 12 as synthetic primers. As the reaction conditions for PCR, the DNA was treated at 94 ° C. for 2 minutes, followed by 30 cycles of denaturation (94 ° C., 15 seconds), annealing (65 ° C., 30 seconds), and extension (68 ° C., 1 minute). . By this PCR, DNA fragments in which EcoR I restriction enzyme recognition sites were added to the 5 'end and 3' end of the DNA sequences encoding hemoglobin alpha and beta chains, respectively, were obtained. DNA fragments amplified by PCR were digested with a restriction enzyme EcoR I (manufactured by Takarashuzo) after phenol extraction and purification by ethanol precipitation. DNA fragments after restriction enzyme treatment were subjected to 2% agarose electrophoresis, resulting in bands corresponding to the respective DNA fragments (DNA fragments encoding hemoglobin α chains; DNA fragments encoding Hbα, SEQ ID NO: 13 and hemoglobin β chains; Hbβ, SEQ ID NO: 14) was cut out and gel extracted (QIAquick Gel Extraction Kit, manufactured by QIAGEN).

(2) linking hemoglobin genes

PAO815 (manufactured by Invitrogen), a shuttle vector of Escherichia coli and yeast, was introduced into E. coli JM109 (manufactured by Takarashuzo) and transformed. The transformant into which pAO815 was introduced was screened in ampicillin addition medium to purify the plasmid from the resulting transformant (QIAprep Spin Miniprep Kit, manufactured by QIAGEN). The plasmid was digested with EcoR I (manufactured by Takarashuzo), double digested by EcoR I and Sal I (manufactured by Takarashuzo), and double digested by BamH I and Bgl II (manufactured by Takarashuzo), A restriction enzyme map was generated to confirm that it was the desired plasmid vector.

Escherichia coli, which was confirmed to have introduced pAO815, was further cultured to purify the plasmid from the grown cells (QIAGEN plasmid Maxi Kit, manufactured by QIAGEN). Purified pAO815 was digested with EcoR I (manufactured by Takarashu-Zhou), followed by dephosphorylation after phenol extraction and purification by ethanol precipitation (Alkaline Phosphatase E. coli C75, manufactured by Takarashu-Zo).

Hbα and Hbβ were ligated to pAO815, respectively, to dephosphorylate (DNA Ligation kit Ver. 1, manufactured by Takarashuzo) to obtain Hbα-pAO815 and Hbβ-pAO815, respectively. The obtained Hbα-pAO815 and Hbβ-pAO815 were introduced into E. coli JM109 (manufactured by Takarashuzo), respectively, and transformed. The transformants into which the target plasmids Hbα-pAO815 and Hbβ-pAO815 were introduced were screened in ampicillin-added medium, and the plasmids were purified from the resulting transformants (QIAprep Spin Miniprep Kit, manufactured by QIAGEN). The plasmid was digested with EcoR I (manufactured by Takarashuzo) and double digested by BamH I and Bgl II (manufactured by Takarashuzo), and a restriction enzyme map was prepared to confirm that it was Hbα-pAO815 and Hbβ-pAO815. .

In addition, the entire sequence of the hemoglobin α chain and the β chain gene is used. For the α chain, the 5 'AOX1 sequencing primer of SEQ ID NO: 15 (manufactured by Invitrogen), the Hbα-sequence primer 1 of SEQ ID NO: 16, and the Hbα-sequence primer of SEQ ID NO: 17 2, and β chains were prepared using ABI Prism 310 Genetic Analyzer (SEQ ID NO: 15) using the 5 'AOX1 sequencing primer of SEQ ID NO: 15 (manufactured by Invitrogen), Hbβ-Sequence Primer 1 of SEQ ID NO: 18, and Hbβ-Sequence Primer 2 of SEQ ID NO: 19. Applied Biosystems).

Escherichia coli, which was confirmed that Hbα-pAO815 and Hbβ-pAO815 had been introduced, was further cultured, and plasmids were purified from the grown cells (QIAGEN plasmid Maxi Kit, manufactured by QIAGEN). Purified Hbα-pAO815 was digested with BamH I and Bgl II (manufactured by Takarashuzo), and the bands corresponding to the Hbα expression cassette were cut out and gel extracted (QIAquick Gel Extraction Kit, manufactured by QIAGEN).

On the other hand, purified Hbβ-pAO815 was digested with BamH I (manufactured by Takarashuzo) without dephosphorylation treatment, purified by phenol extraction and ethanol precipitation.

The Hbα expression cassette was ligated to Hbβ-pAO815 enzymatically digested with BamH I (DNA Ligation kit Ver. 1, manufactured by Takarashuzo) to obtain Hbβ-Hbα-pAO815. The obtained Hbβ-Hbα-pAO815 was introduced into E. coli DH5α (Toyo Boseki) for transformation. The transformant into which the target plasmid Hbβ-Hbα-pAO815 was introduced was screened in an ampicillin addition medium to purify the plasmid from the resulting transformant (QIAprep Spin Miniprep Kit, produced by QIAGEN). The plasmid was digested with EcoR I (manufactured by Takarashuzo) and double digested by BamH I and Bgl II (manufactured by Takarashuzo), and a restriction enzyme map was prepared to confirm that it was Hbβ-Hbα-pAO815. 2 shows the steps up to the construction of Hbβ-Hbα-pAO815.

(3) expression of hemoglobin

Hbβ-Hbα-pAO815 was digested with the restriction enzyme Sal I, purified by phenol extraction and ethanol precipitation, followed by Pichia yeast (GS115 strain) using an electroporation device (Gene Pulser II Electroporation System, manufactured by BIO-RAD). Transformation was performed by homologous recombination into the HIS4 locus (see FIG. 3). The resulting transformants were cultured in BMMY liquid medium to confirm hemoglobin expression, and then glycerol stock was obtained.

(4) purification of hemoglobin

The transformed Pichia yeast was incubated for 48 hours in BMGY liquid medium, and then for 96 hours with 1% methanol added every 12 hours in BMMY medium. The yeast was precipitated by centrifugation (6,000 g × 10 min), resuspended in crushing buffer, and glass beads (425-600 μm) were added and vigorously stirred to break up the yeast.

The supernatant was recovered by centrifuging the crushed cells at 10,000 g for 30 minutes. This was dialyzed with 10 mM tris hydrochloric acid buffer (pH 7.5), passed through a Q-Sepharose ^™ Fast Flow column (manufactured by Amersham Biosciences), and bound to a Q-Sepharose ^™ Fast Flow column with a pH of 8.5. , Hemoglobin was eluted by a concentration gradient of 0 → 300 mM NaCl. It was exchanged with 200 mM sodium acetate buffer (pH 5.5), and it couple | bonded with the Blue Sepharose CL-6B column (made by Amersham Biosciences), and hemoglobin was eluted by the concentration gradient of 0-> 3 M NaCl. The eluate was then dialyzed with 0.5 M ammonium sulfate / 100 mM sodium phosphate buffer (pH 7.0), and then bound to a HiTrap Butyl FF column (manufactured by Amersham Biosciences) to elute hemoglobin with 70% ethanol. . Finally, CO was blown in and stored at 4 ° C.

(5) Confirmation of hemoglobin

The final sample was subjected to SDS polyacrylamide gel electrophoresis to detect a single band with an expected molecular weight of about 15,000. In addition, the N-terminal amino acids were analyzed and found to be completely consistent with the amino acid sequence of hemoglobin derived from human blood. The molecular weights of the α chain and the β chain were calculated by MALDI TOF-MS to be 15,122 and 15,866, respectively, and were consistent with the calculated values.

The production method of the present invention is very useful in that human hemoglobin, which can be safely administered to a living body, can be supplied in large quantities easily because there is no risk of contamination such as viruses.

While the invention has been described with emphasis on preferred forms, it will be apparent to those skilled in the art that the preferred forms may be altered. The present invention is intended that the present invention may be practiced by methods other than those described in detail herein. Accordingly, the present invention is intended to embrace all such changes as fall within the spirit and scope of the appended claims.

In addition, the contents of all the publications including the patents and patent application specifications described herein are incorporated herein to the extent that they are hereby incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS It is explanatory drawing which shows the principle which connects the expression cassette of hemoglobin alpha chain, and the expression cassette of beta chain.

FIG. 2 is an explanatory diagram showing a schematic procedure in the production of hemoglobin in Examples. FIG.

It is explanatory drawing which shows the recombination method of the hemoglobin to the Pichia chromosome in an Example.

                         SEQUENCE LISTING <110> Nipro Corporation   <120> Production of Recombinant Human Hemoglobin Using Pichia Yeast <130> A7594 <160> 19 <170> PatentIn version 3.3 <210> 1 <211> 426 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (426) <400> 1 atg gtt ctg tct ccg gct gac aaa act aac gtt aaa gct gct tgg ggt 48 Met Val Leu Ser Pro Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly 1 5 10 15 aaa gtt ggt gct cat gct ggt gaa tac ggt gct gaa gct ctg gaa cgt 96 Lys Val Gly Ala His Ala Gly Glu Tyr Gly Ala Glu Ala Leu Glu Arg             20 25 30 atg ttc ctg tct ttc ccg act act aaa act tac ttc ccg cat ttc gac 144 Met Phe Leu Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp         35 40 45 ctg tct cat ggt tct gct cag gtt aaa ggt cat ggt aaa aaa gtt gct 192 Leu Ser His Gly Ser Ala Gln Val Lys Gly His Gly Lys Lys Val Ala     50 55 60 gac gct ctg act aac gct gtt gct cat gtt gac gac atg ccg aac gct 240 Asp Ala Leu Thr Asn Ala Val Ala His Val Asp Asp Met Pro Asn Ala 65 70 75 80 ctg tct gct ctg tct gac ctg cat gct cat aaa ctg cgt gtt gac ccg 288 Leu Ser Ala Leu Ser Asp Leu His Ala His Lys Leu Arg Val Asp Pro                 85 90 95 gtt aac ttc aaa ctg ctg tct cat tgc ctg ctg gtt act ctg gct gct 336 Val Asn Phe Lys Leu Leu Ser His Cys Leu Leu Val Thr Leu Ala Ala             100 105 110 cat ctg ccg gct gag ttt act ccg gct gtt cat gct tct ctg gac aaa 384 His Leu Pro Ala Glu Phe Thr Pro Ala Val His Ala Ser Leu Asp Lys         115 120 125 ttc ctg gct tct gtt tct act gtt ctg act tct aaa tac cgt 426 Phe Leu Ala Ser Val Ser Thr Val Leu Thr Ser Lys Tyr Arg     130 135 140 <210> 2 <211> 142 <212> PRT <213> Homo sapiens <400> 2 Met Val Leu Ser Pro Ala Asp Lys Thr Asn Val Lys Ala Ala Trp Gly 1 5 10 15 Lys Val Gly Ala His Ala Gly Glu Tyr Gly Ala Glu Ala Leu Glu Arg             20 25 30 Met Phe Leu Ser Phe Pro Thr Thr Lys Thr Tyr Phe Pro His Phe Asp         35 40 45 Leu Ser His Gly Ser Ala Gln Val Lys Gly His Gly Lys Lys Val Ala     50 55 60 Asp Ala Leu Thr Asn Ala Val Ala His Val Asp Asp Met Pro Asn Ala 65 70 75 80 Leu Ser Ala Leu Ser Asp Leu His Ala His Lys Leu Arg Val Asp Pro                 85 90 95 Val Asn Phe Lys Leu Leu Ser His Cys Leu Leu Val Thr Leu Ala Ala             100 105 110 His Leu Pro Ala Glu Phe Thr Pro Ala Val His Ala Ser Leu Asp Lys         115 120 125 Phe Leu Ala Ser Val Ser Thr Val Leu Thr Ser Lys Tyr Arg     130 135 140 <210> 3 <211> 441 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (441) <400> 3 atg gtt cat ctg act ccg gaa gaa aaa tct gct gtt act gct ctg tgg 48 Met Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp 1 5 10 15 ggt aaa gtt aac gtt gac gaa gtt ggt ggt gaa gct ctg ggt cgt ctg 96 Gly Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu             20 25 30 ctg gtt gtt tac ccg tgg act cag cgt ttc ttc gaa tct ttc ggt gac 144 Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp         35 40 45 ctg tct act ccg gac gct gtt atg ggt aac ccg aaa gtt aaa gct cat 192 Leu Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His     50 55 60 ggt aaa aaa gtt ctg ggt gct ttc tct gac ggt ctg gct cat ctg gac 240 Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp 65 70 75 80 aac ctg aaa ggt act ttc gct act ctg tct gaa ctg cat tgc gac aaa 288 Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys                 85 90 95 ctg cat gtt gac ccg gaa aac ttc cgt ctg ctg ggt aac gtt ctg gtt 336 Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val             100 105 110 tgc gtt ctg gct cat cat ttc ggt aaa gag ttt act ccg ccg gtt cag 384 Cys Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln         115 120 125 gct gct tac cag aaa gtt gtt gct ggt gtt gct aac gct ctg gct cat 432 Ala Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His     130 135 140 aaa tac cat 441 Lys Tyr His 145 <210> 4 <211> 147 <212> PRT <213> Homo sapiens <400> 4 Met Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp 1 5 10 15 Gly Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala Leu Gly Arg Leu             20 25 30 Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp         35 40 45 Leu Ser Thr Pro Asp Ala Val Met Gly Asn Pro Lys Val Lys Ala His     50 55 60 Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp 65 70 75 80 Asn Leu Lys Gly Thr Phe Ala Thr Leu Ser Glu Leu His Cys Asp Lys                 85 90 95 Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val             100 105 110 Cys Val Leu Ala His His Phe Gly Lys Glu Phe Thr Pro Pro Val Gln         115 120 125 Ala Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Ala His     130 135 140 Lys Tyr His 145 <210> 5 <211> 30 <212> DNA <213> Artificial <220> <223> Primer <400> 5 gccggctgag tttactccgg ctgttcatgc 30 <210> 6 <211> 30 <212> DNA <213> Artificial <220> <223> Primer <400> 6 gcatgaacag ccggagtaaa ctcagccggc 30 <210> 7 <211> 30 <212> DNA <213> Artificial <220> <223> Primer <400> 7 cggtaaagag tttactccgc cggttcaggc 30 <210> 8 <211> 30 <212> DNA <213> Artificial <220> <223> Primer <400> 8 gcctgaaccg gcggagtaaa ctctttaccg 30 <210> 9 <211> 34 <212> DNA <213> Artificial <220> <223> Primer <400> 9 gcttaaaagg agatgaattc atggttctgt ctcc 34 <210> 10 <211> 34 <212> DNA <213> Artificial <220> <223> Primer <400> 10 cgggctttgt tagcagccga attcctatta acgg 34 <210> 11 <211> 34 <212> DNA <213> Artificial <220> <223> Primer <400> 11 ggagatgaat tcatggttca tctgactccg gaag 34 <210> 12 <211> 40 <212> DNA <213> Artificial <220> <223> Primer <400> 12 ccttttgaat tcctattaat ggtatttatg agccagagcg 40 <210> 13 <211> 444 <212> DNA <213> Artificial <220> <223> Hb „A cDNA having EcoR I sites at 5 'and 3' ends <400> 13 gaattcatgg ttctgtctcc ggctgacaaa actaacgtta aagctgcttg gggtaaagtt 60 ggtgctcatg ctggtgaata cggtgctgaa gctctggaac gtatgttcct gtctttcccg 120 actactaaaa cttacttccc gcatttcgac ctgtctcatg gttctgctca ggttaaaggt 180 catggtaaaa aagttgctga cgctctgact aacgctgttg ctcatgttga cgacatgccg 240 aacgctctgt ctgctctgtc tgacctgcat gctcataaac tgcgtgttga cccggttaac 300 ttcaaactgc tgtctcattg cctgctggtt actctggctg ctcatctgcc ggctgagttt 360 actccggctg ttcatgcttc tctggacaaa ttcctggctt ctgtttctac tgttctgact 420 tctaaatacc gttaatagga attc 444 <210> 14 <211> 459 <212> DNA <213> Artificial <220> <223> Hb „A cDNA having EcoR I sites at 5 'and 3' ends <400> 14 gaattcatgg ttcatctgac tccggaagaa aaatctgctg ttactgctct gtggggtaaa 60 gttaacgttg acgaagttgg tggtgaagct ctgggtcgtc tgctggttgt ttacccgtgg 120 actcagcgtt tcttcgaatc tttcggtgac ctgtctactc cggacgctgt tatgggtaac 180 ccgaaagtta aagctcatgg taaaaaagtt ctgggtgctt tctctgacgg tctggctcat 240 ctggacaacc tgaaaggtac tttcgctact ctgtctgaac tgcattgcga caaactgcat 300 gttgacccgg aaaacttccg tctgctgggt aacgttctgg tttgcgttct ggctcatcat 360 ttcggtaaag agtttactcc gccggttcag gctgcttacc agaaagttgt tgctggtgtt 420 gctaacgctc tggctcataa ataccattaa taggaattc 459 <210> 15 <211> 21 <212> DNA <213> Artificial <220> <223> Primer <400> 15 gactggttcc aattgacaag c 21 <210> 16 <211> 30 <212> DNA <213> Artificial <220> <223> Primer <400> 16 cggttaactt caaactgctg tctcattgcc 30 <210> 17 <211> 23 <212> DNA <213> Artificial <220> <223> Primer <400> 17 gccaggaatt tgtccagaga agc 23 <210> 18 <211> 23 <212> DNA <213> Artificial <220> <223> Primer <400> 18 ccgtctgctg ggtaacgttc tgg 23 <210> 19 <211> 24 <212> DNA <213> Artificial <220> <223> Primer <400> 19 gcaacaccag caacaacttt ctgg 24  

Claims (9)

Expression vectors containing DNA encoding human hemoglobin α chains under control of a promoter capable of functioning in Pichia yeast, and DNA encoding human hemoglobin β chains under control of a promoter capable of functioning in Pichia yeast A method for producing a functional recombinant human hemoglobin, comprising culturing Pichia yeast transformed with an expression vector containing in a medium and recovering human hemoglobin from the culture obtained. The method of claim 1, wherein the DNA encoding the human hemoglobin α chain and the DNA encoding the human hemoglobin β chain are on the same vector. The method of claim 2, wherein the DNA encoding the human hemoglobin β chain is upstream in the transcription direction. The method of claim 1, wherein the DNA encoding the human hemoglobin α chain and the DNA encoding the human hemoglobin β chain are under the control of separate promoters. 5. The method of claim 1, wherein the DNA encoding the human hemoglobin α chain and the DNA encoding the human hemoglobin β chain are under control of an alcohol oxidase 1 promoter. 6. The method of claim 5, wherein the transformed Pichia yeast is cultivated in a liquid medium having methanol as a single carbon source. The method of claim 6, wherein the culture is a fed-batch culture. 8. The method of claim 1, wherein the Pichia yeast is Pichia pastoris . 9. From the upstream side of the transcription direction, alcohol oxidase 1 promoter, DNA encoding human hemoglobin β chain, terminator capable of functioning in Pichia yeast, alcohol oxidase 1 promoter, DNA encoding human hemoglobin α chain and Pichia yeast Expression vector containing in order of terminator that can function in.

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