Method for producing recombinant proteins using PDI from trichoderma strains

Abstract

The present invention relates to a method for producing a recombinant protein by using PDI from a Trichoderma strain. The variant yeast comprising a gene encoding PDI from a Trichoderma genus strain of the present invention highly expresses a target protein, and thus is useful for producing a target recombinant protein at high yield.

Description

Technical Field

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0175628, filed on December 15, 2022, and the entire content disclosed in the document of the corresponding Korean patent application is incorporated as part of this specification.

[0003] The present invention relates to a method for producing a recombinant protein using PDI from a Trichoderma sp. strain. Background Art

[0004] Host cells mainly used for producing recombinant proteins include Escherichia coli, yeast (Saccharomyces cerevisiae), and animal cells such as CHO cells. Among them, yeast has several advantages compared to Escherichia coli. Yeast is a eukaryotic cell that is genetically identical to higher organisms (different from Escherichia coli), and has a transcription and translation system similar to genes from higher organisms, and can remove introns by splicing, and has a secretory organ similar to the Golgi apparatus of higher organisms. Therefore, it can produce and secrete proteins activated by post-translational modification. In addition, compared to Escherichia coli, yeast has the advantage that it can secrete and produce recombinant proteins more effectively extracellularly, which helps in the separation and purification of the protein, and when producing a protein in yeast, there are no problems caused by endotoxins (in the case of pharmaceutical proteins, endotoxins should be removed). Among these yeasts, Saccharomyces cerevisiae is representatively used, and since the production of recombinant proteins using Saccharomyces cerevisiae was reported in 1981, Saccharomyces cerevisiae has been actively studied by many researchers. However, in order to obtain a high expression rate in Saccharomyces cerevisiae, multi-copy plasmids are required, and when performing high-concentration cell culture in a large-scale fermenter, the distribution, copy number, stability, etc. of these plasmids often become problems, and most promoters from glycolytic genes are constitutive promoters. Therefore, when using them to express proteins, there is a problem that cells that have lost the plasmid may be dominant, and there is also a problem that promoters that can be controlled are difficult to precisely control. As an alternative to solve these problems, a facultative methylotrophic yeast, Pichia pastoris, has recently attracted attention. Pichia pastoris is a methylotrophic yeast, and foreign genes can be integrated into chromosomal DNA, and AOX1, which is the promoter of an enzyme in the methanol-utilizing pathway, is used, and this promoter has the very strong advantage.

[0005] Protein disulfide isomerase (PDI) is an enzyme that catalyzes the thiol:disulfide exchange reaction. It resides in the endoplasmic reticulum, is newly synthesized, and transfers disulfide bonds to secreted proteins entering the endoplasmic reticulum. PDI is a redox enzyme that not only has the activity of transferring disulfide bonds but also has isomerization activity to break incorrectly linked disulfide bonds and convert them into structurally stable disulfide bonds. When PDI functions abnormally, the substrate is degraded rather than leaving the endoplasmic reticulum and being secreted into the Golgi apparatus or extracellularly. Therefore, depending on the activity level of PDI, the synthesis efficiency of secreted proteins may vary.

[0006] Therefore, attempts have been made to use PDI to produce recombinant proteins. It has been reported that fusing PDI from a thermophilic fungus to the amino terminus of the target protein and secreting it extracellularly in a Bacillus brevis strain (Kajino, T. et al. (2000) Appl. Environ. Microbiol. 66:638 - 642), or co - expressing dsbC (a type of PDI) and the target protein in the oxidative cytoplasm of mutant Escherichia coli (Bessette, P. H. et al. (1999) Proc. Natl. Acad. Sci. USA, 96:13703 - 13708), etc. However, it is known that when secreted extracellularly from a Bacillus strain, due to problems such as protease secretion in the Bacillus strain, there is a significant drawback of a very low yield of the target protein, and when dsbC and the target protein are co - expressed in the cytoplasm, the expression rate is low. Summary of the Invention

[0007] Technical Problem

[0008] One embodiment of the present application provides a method for producing a recombinant protein, comprising:

[0009] 1) Preparing a mutant yeast, including performing the following steps i) and ii) simultaneously, sequentially, or in the reverse order:

[0010] i) Introducing a vector containing a gene encoding PDI (protein disulfide isomerase) from a Trichoderma strain into the yeast;

[0011] ii) Introducing a vector containing a gene encoding the target protein into the yeast; and

[0012] 2) Culturing the mutant yeast prepared in step 1).

[0013] Another embodiment of the present application provides a mutant yeast for producing a target protein, which contains a gene encoding PDI (protein disulfide isomerase) from a Trichoderma strain.

[0014] Other embodiments of the present application provide compositions for producing a target protein in mutant yeast, which comprise a gene encoding PDI (protein disulfide isomerase) from a Trichoderma strain or a vector containing the same.

[0015] Technical solution

[0016] To achieve the above object, the present invention provides a mutant yeast strain transformed with a gene encoding PDI (protein disulfide isomerase) from a Trichoderma strain, and a method for producing a target protein using the mutant yeast strain.

[0017] Hereinafter, the present invention will be described in more detail.

[0018] The present invention provides a method for producing a recombinant protein, comprising:

[0019] 1) Preparing mutant yeast, including performing the following steps i) and ii) simultaneously, sequentially or in reverse order:

[0020] i) Introducing a vector containing a gene encoding PDI (protein disulfide isomerase) from a Trichoderma strain into yeast;

[0021] ii) Introducing a vector containing a gene encoding the target protein into yeast; and

[0022] 2) Culturing the mutant yeast prepared in step 1).

[0023] The term "PDI (protein disulfide isomerase)" used in this specification refers to an enzyme that catalyzes the thiol:disulfide exchange reaction and can be used interchangeably with "protein disulfide isomerase".

[0024] The PDI in step i) of step 1) above can be PDI from a Trichoderma strain, and preferably, can be PDI from a Trichoderma reesei strain.

[0025] In one embodiment of the present invention, the PDI may comprise the amino acid sequence of SEQ ID NO:14, or consist of the amino acid sequence of SEQ ID NO:14, and polypeptides having an amino acid sequence in which a part of the amino acid sequence of SEQ ID NO:14 is deleted, modified, substituted or added may also be included within the scope of the present application, as long as it has the same or corresponding enzyme activity as PDI. In addition, regardless of the source of the microorganism, polypeptides having the same or corresponding conversion activity as PDI, which are polypeptides having at least 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 92% or more, 94% or more, 96% or more, 98% or more, or 99% or more, 99.5% or more, or 99.8% or more homology or identity with the amino acid sequence of SEQ ID NO:14, may also be included as PDI within the scope of the present application.

[0026] The yeast in step i) and step ii) of the above step 1) may be any one selected from the group consisting of strains of the genus Pichia, Candida, and Hansenula. In one embodiment, the yeast may be Pichia pastoris, and more specifically, it may be Pichia pastoris strain BG16, but is not limited thereto.

[0027] The target protein in step ii) of step 1) may be a protein containing a disulfide bond in the protein, or a protein containing at least 3 disulfide bonds in the protein, but is not limited thereto.

[0028] In one embodiment of the present invention, the target protein may be any one or more selected from the group consisting of transferrin, albumin, and proinsulin, and transferrin, albumin, and proinsulin may be bovine-derived proteins, but are not limited thereto.

[0029] In the present specification, the term "transferrin" may refer to serum transferrin, and it may be used as one of the factors to replace serum (FBS, fetal bovine serum) in a serum-free medium (FBS-free medium), etc. In one embodiment, the transferrin may comprise the following amino acid sequence of SEQ ID NO:6, or consist of the amino acid sequence of SEQ ID NO:6, but is not limited thereto.

[0030] [SEQ ID NO:6] Bovine transferrin (N495Q is in bold)

[0031] DPERTVRWCTISTHEANKCASFRENVLRILESGPFVSCVKKTSHMDCIKAISNNEADAVTLDGGLVYEAGLKPNNLKPVVAEFHGTKDNPQTHYYAVAVVKKDTDFKLNELRGKKSCHTGLGRSAGWNIPMGKLYKELPDPQESIQRAAANFFSASCVPCADQSSFPKLCQLCAGKGTDKCACSNHEPYFGYSGAFKCLMEGAGDVAFVKHSTVFDNLPNPEDRKNYELLCGDNTRKSVDDYQECYLAMVPSHAVVARTVGGKEDVIWELLNHAQEHFGKDKPDNFQLFQSPHGKDLLFKDSADGFLKIPSKMDFELYLGYEYVTALQNLRESKPPDSSKDECMVKWCAIGHQERTKCDRWSGFSGGAIECETAENTEECIAKIMKGEADAMSLDGGYLYIAGKCGLVPVLAENYKTEGESCKNTPEKGYLAVAVVKTSDANINWNNLKDKKSCHTAVDRTAGWNIPMGLLYSKINNCKFDEFFSAGCAPGSPRQSSLCALCIGSEKGTGKECVPNSNERYYGYTGAFRCLVEKGDVAFVKDQTVIQNTDGNNNEAWAKNLKKENFEVLCKDGTRKPVTDAENCHLARGPNHAVVSRKDKATCVEKILNKQQDDFGKSVTDCTSNFCLFQSNSKDLLFRDDTKCLASIAKKTYDSYLGDDYVRAMTNLRQCSTSKLLEACTFHKP

[0032] In the present invention, the term "albumin" may refer to serum albumin, and it can be used as one of the factors to replace serum in serum-free media and the like. In one embodiment, the albumin may comprise the amino acid sequence of SEQ ID NO:24 below, or consist of the amino acid sequence of SEQ ID NO:24, but is not limited thereto.

[0033] [SEQ ID NO:24]

[0034] DTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFDEHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEPERNECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELLYYANKYNGVFQECCQAEDKGACLLPKIETMREKVLASSARQRLRCASIQKFGERALKAWSVARLSQKFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKECCDKPLLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKQIKKQTALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVVSTQTALA

[0035] In the present specification, the term "precursor insulin" can be used as one of the factors to replace serum in a serum-free medium or the like. In one embodiment, the precursor insulin may comprise the following amino acid sequence of SEQ ID NO:30, or consist of the amino acid sequence of SEQ ID NO:30, but is not limited thereto.

[0036] [SEQ ID NO:30] The spacer sequence is underlined

[0037] EPK FVNQHLCGSHLVEALYLVCGERGFFYTPKA AAK GIVEQCCASVCSLYQLENYCN

[0038] In this specification, the term "vector" may include a DNA construct that contains a base sequence of a polynucleotide operably linked to a suitable expression regulatory region (or expression regulatory sequence) so as to express, in a suitable host, a polynucleotide encoding PDI from a Trichoderma strain or a polynucleotide encoding a target protein. The expression regulatory region may include a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating transcription termination and termination. After being transformed into a suitable host cell, the vector may replicate or function independently of the host genome and may be integrated into the genome itself.

[0039] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors may include natural or recombinant plasmids, cosmids, viruses, and phages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. may be used as phage vectors or cosmid vectors, and vectors based on pDZ, based on pBR, based on pUC, based on pBluescriptII, based on pGEM, based on pTZ, based on pCL, and based on pET, etc. may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pPICZαA vectors, etc. may be used.

[0040] As an example, a polynucleotide encoding PDI from a Trichoderma strain or a polynucleotide encoding a target protein may be inserted into a chromosome by a vector for inserting into a chromosome in a cell. The polynucleotide may be inserted into the chromosome by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker for confirming whether the polynucleotide is inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, that is, to confirm the insertion of the target nucleic acid molecule, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, cytotoxic agent resistance, or surface polypeptide expression may be used. In an environment treated with a selection agent, only cells expressing the selection marker survive or exhibit additional phenotypic traits, thereby enabling the selection of transformed cells.

[0041] In the present application, the term "transformation" refers to the introduction of a vector containing a specific polynucleotide into a host cell or microorganism such that a polypeptide encoded by the polynucleotide can be expressed in the host cell. As long as the transformed polynucleotide can be expressed in the host cell, the host cell can include all of these, regardless of whether they are inserted and located in the chromosome of the host cell or located extrachromosomally. In addition, the polynucleotide contains DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all the elements required for its own expression. The expression cassette typically may contain a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette can be in the form of an expression vector capable of self-replication. In addition, the polynucleotide is introduced into the host cell in its own form and operably linked to the sequences required for expression in the host cell, but is not limited thereto.

[0042] In addition, the above term "operably linked" means that a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding PDI from a Trichoderma strain or the target protein of the present application is functionally linked to the polynucleotide sequence.

[0043] In the present specification, the term "culturing" refers to culturing the mutant yeast of the present application under approximately controlled environmental conditions. The culturing process of the present application can be carried out according to suitable culture media and culture conditions known in the art. Those skilled in the art can easily adjust and use such a culturing method according to the selected strain. Specifically, the culturing can be carried out in a batch, continuous, and / or fed-batch manner, but is not limited thereto.

[0044] In the present specification, the term "culture medium" refers to a substance that mixes the nutrients required for culturing the mutant yeast of the present application as the main components, and provides nutrients, growth factors, etc., including water that is indispensable for survival and growth. Specifically, there is no particular limitation on any culture medium used for culturing ordinary microorganisms for the culture medium and other culture conditions for culturing the mutant yeast of the present application, but the mutant yeast of the present application can be cultured in an aerobic condition in a normal culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin, etc., while adjusting the temperature, pH, etc. Specifically, the culture media for yeast can be found in documents such as ["Yeast", Current Protocols in Molecular Biology, 2017], ["Pichia Protocols", Methods in Molecular Biology, 2007], etc.

[0045] Carbon sources may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc.; methanol or ethanol, etc. In addition, natural organic nutrients such as starch hydrolysis products, molasses, blackstrap molasses, rice bran, cassava, cane molasses, and corn steep liquor can be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted with reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in different ways without limitation. These carbon sources can be used alone or in combination of at least two, but not limited thereto.

[0046] As nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium citrate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc. can be used; organic nitrogen sources such as amino acids (including glutamate, methionine, glutamine, etc.), peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources can be used alone or in combination of at least two, but not limited thereto.

[0047] As phosphorus sources, it may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate or their corresponding sodium-containing salts, etc. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. can be used, and other amino acids, vitamins and / or appropriate precursors, etc. can be included. These components or precursors can be added to the culture medium in a batch or continuous manner. However, it is not limited thereto.

[0048] In addition, during the process of culturing the mutant yeast of the present application, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphate, sulfate, etc. to the culture medium in an appropriate method. In addition, during the culturing process, an antifoaming agent such as fatty acid polyethylene glycol ester can be used to inhibit the generation of bubbles. In addition, in order to maintain the aerobic conditions of the culture medium, oxygen or oxygen-containing gas can be injected into the culture medium, or in order to maintain anaerobic and microaerophilic conditions, no gas is injected, or nitrogen, oxygen or carbon dioxide gas can be injected, but not limited thereto.

[0049] In the culturing step of the present invention, the culturing temperature can be maintained at 20°C to 45°C, specifically, 25°C to 40°C, and the culturing can be carried out for about 10 to 160 hours, but not limited thereto.

[0050] The recombinant protein produced by the culturing of the present invention can be secreted into the culture medium or retained in the cells.

[0051] The method for producing a recombinant protein of the present invention may further include the step of preparing the mutant yeast of the present invention, the step of preparing a culture medium for culturing the mutant yeast, or a combination thereof (in any order), for example, before the culturing step.

[0052] The method for producing a recombinant protein of the present invention may further include recovering the recombinant protein from the culture medium according to which the culture is carried out (the culture medium for carrying out the culture) or the mutant yeast. A recovery step may be further included after the culturing step.

[0053] The recovery may be carried out according to the method for culturing the mutant yeast of the present invention, and the target protein may be collected using appropriate methods known in the art, such as batch, continuous, or fed-batch culture methods, etc. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, sonication, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, or a combination of these methods may be used, and the target protein may be recovered from the culture medium or the mutant yeast using appropriate methods known in the art.

[0054] In addition, the method for producing the recombinant protein of the present invention may further include a purification step. The purification may be carried out using appropriate methods known in the art. In one embodiment, when the method for producing the recombinant protein of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out discontinuously (or continuously) in any order, or may be carried out simultaneously or integrated into one step, but is not limited thereto.

[0055] In addition, the present invention provides a mutant yeast for producing a target protein, which contains a gene encoding PDI (protein disulfide isomerase) from a Trichoderma strain.

[0056] The PDI, target protein, and yeast from the Trichoderma strain are the same as those described above.

[0057] The mutant yeast may be a yeast in which the gene encoding PDI from a Trichoderma strain is inserted or integrated into the yeast genome, or may be a yeast in which the gene encoding PDI from a Trichoderma strain exists outside the yeast genome.

[0058] Compared with the yeast without the introduction, due to the introduction of the gene encoding PDI from a heterologous Trichoderma strain, the mutant yeast may have an increased productivity of the target protein.

[0059] As an example, compared to the target protein productivity of the parental strain before mutation or an unmodified microorganism, a mutant yeast with increased target protein productivity can have an increased target protein productivity of about 1% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 26% or more, about 27% or more, about 28% or more, about 29% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, about 35% or more, about 36% or more, about 37% or more, about 38% or more, about 39% or more, about 40% or more, about 41% or more, about 42% or more, about 43% or more, about 44% or more, about 45% or more, about 46% or more, about 47% or more, about 48% or more, about 49% or more, about 50% or more (the upper limit is not particularly limited. For example, it can be about 200% or less, about 150% or less, about 100% or less, or about 50% or less).

[0060] In another embodiment, compared to the parental strain before mutation or an unmodified microorganism, a mutant yeast with increased productivity can have an increased target protein productivity (or production capacity, or yield) of about 1.1-fold or more, about 1.12-fold or more, about 1.13-fold or more, 1.15-fold or more, 1.16-fold or more, 1.17-fold or more, 1.18-fold or more, 1.19-fold or more, about 1.2-fold or more, 1.25-fold or more, about 1.3-fold or more, about 1.4-fold or more, or about 1.5-fold or more (the upper limit is not particularly limited. For example, it can be about 10-fold or less, about 5-fold or less, about 3-fold or less, or about 2-fold or less), but is not limited thereto. The term "about" encompasses the entire range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., which includes all values equal to or similar to the value after the term "about", but is not limited thereto.

[0061] In this specification, the term "unmodified microorganism" does not exclude strains that may have mutations naturally occurring in the microorganism. It can refer to the wild-type strain or the natural strain itself, or the strain before its traits are changed due to genetic mutations caused by natural or artificial factors. For example, an unmodified microorganism can refer to a strain in which the gene of PDI from the Trichoderma strains described in this specification has not been introduced or the strain before the introduction of the gene. "Unmodified microorganism" can be used interchangeably with "strain before modification", "microorganism before modification", "unmutated strain", "unmodified strain", "unmutated microorganism", or "reference microorganism". In one embodiment, the "unmodified microorganism" can be a Pichia pastoris strain, more specifically, the Pichia pastoris BG16 strain, but is not limited thereto.

[0062] In addition, the present invention provides a composition for producing a target protein in mutant yeast, which comprises a gene encoding PDI (protein disulfide isomerase) from a Trichoderma strain or a recombinant vector containing the same.

[0063] The PDI, target protein, and yeast from the Trichoderma strain are the same as those described above.

[0064] In one embodiment, the composition for production may further comprise any suitable excipient commonly used in compositions for producing a target protein, and such excipient may be, for example, a preservative, wetting agent, dispersant, suspending agent, buffer, stabilizer, or tonicity agent, but is not limited thereto.

[0065] In addition, the present invention provides a method for increasing the target protein productivity of a microorganism or for conferring target protein productivity to a microorganism, comprising introducing (e.g., transforming) a polynucleotide encoding PDI (protein disulfide isomerase) from a Trichoderma strain and / or a recombinant vector containing the polynucleotide.

[0066] Beneficial effects

[0067] The mutant yeast of the present invention containing the gene encoding PDI from a Trichoderma strain highly expresses the target protein, and thus the target recombinant protein can be produced in high yield using the same. Brief description of the drawings

[0068] Figure 1 It is a figure showing a schematic diagram of the pPICZFRT recombinant plasmid.

[0069] Figure 2 It is a figure showing a schematic diagram of the pPICZFRT-bTRANSFERRIN (N495Q) recombinant plasmid.

[0070] Figure 3 It is a figure showing the SDS-PAGE results of the transferrin expression levels in the culture supernatants for comparing the Pichia pastoris PDI-introduced strain (CF04-1024), the Ogataea angusta PDI-introduced strain (CF04-1025), and the Trichoderma reesei PDI-introduced strain (CF04-1026).

[0071] Figure 4 It is a figure showing a schematic diagram of the pPICZFRT-PDI(TR) recombinant plasmid.

[0072] Figure 5This is a figure showing the SDS-PAGE results for comparing the albumin expression levels in the culture supernatants of the strain (CF04-1005) in which the gene encoding bovine albumin was introduced into the Pichia pastoris BG16 strain and the strain (CF04-1038) in which the gene was introduced into CF04-1035.

[0073] Figure 6 This is a figure showing the SDS-PAGE results for comparing the preproinsulin expression levels in the culture supernatants of the strain (CF04-1017) in which the gene encoding preproinsulin was introduced into the Pichia pastoris BG16 strain and the strain (CF04-1040) in which the gene was introduced into CF04-1035. Detailed implementation mode

[0074] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are intended to illustratively describe at least one specific implementation, and the scope of the present invention is not limited by these examples.

[0075] Example 1. Preparation of pPICZFRT plasmid

[0076] A vector for transforming Pichia pastoris was prepared by the following method.

[0077] DNA (SEQ ID NO:1) consisting of an FRT site, a restriction enzyme recognition site for cloning, an LRA3 (L-rhamnonic acid dehydratase) promoter expressed under rhamnose induction conditions, and a Flp recombinase was synthesized to ligate the 1341-3593 bp region of the pPICZαA vector (Invitrogen TM , catalog number: V19520). Using the DNA of SEQ ID NO:1 synthesized for such gene manipulation as a template, and using primers having the base sequences of SEQ ID NO:2 and SEQ ID NO:3, the region of SEQ ID NO:1 was amplified by PCR. Using the pPICZαA vector DNA as a template, and using primers having the base sequences of SEQ ID NO:4 and SEQ ID NO:5, the 1341-3593 bp region of the pPICZαA vector was amplified by PCR. For the PCR reaction, Pfu-X DNA polymerase (Solg TM , catalog number: SPX16-R500) was used, and the manufacturer's protocol was followed. The two amplified fragments were cloned using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method to obtain a recombinant plasmid, which was named pPICZFRT (seeFigure 1 )。

[0078] Example 2. Preparation of a Pichia pastoris strain expressing non-glycosylated bovine transferrin

[0079] As the amino acid sequence of bovine transferrin, the sequence at positions 19 to 704 except for the signal sequence in NCBI reference sequence ID: NP_803450.2 was used. In this sequence, in order to remove glycosylation, the N (asparagine) at position 495 was replaced with Q (glutamine), and this sequence was used in a non-glycosylated form. The amino acid sequence of non-glycosylated bovine transferrin is shown in SEQ ID NO: 6.

[0080] In order to express a protein with the amino acid sequence of SEQ ID NO: 6 in Pichia pastoris, the codons of the DNA were optimized. The base sequence of the DNA codon optimization is shown in SEQ ID NO: 7.

[0081] In front of the base sequence of SEQ ID NO: 7, the AOX1 promoter for methanol-induced expression was ligated, followed by the α-mating factor sequence for secreted expression, and the AOX1 transcription terminator was ligated at the back, and a DNA fragment was generated by gene synthesis. Using primers with SEQ ID NO: 8 and SEQ ID NO: 9, and using Pfu-X DNA polymerase, the fragment generated was amplified by PCR reaction, and pPICZFRT was cut with KpnI-HF (NEB, catalog number: R3142) and NotI-HF (NEB, catalog number: R3189). The two fragments obtained in this way were cloned using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method to obtain a recombinant plasmid, which was named pPICZFRT-bTRANSFERRIN (N495Q) (see Figure 2 )。

[0082] In order to introduce the pPICZFRT-bTRANSFERRIN (N495Q) vector into a Pichia pastoris strain and transform it, the AOX1 promoter site was linearized using PmeI (NEB, catalog number: R0560S). After that, Expin TMThe PCR SV kit (GeneAll, catalog number: 103-102) purifies the linearized vector. As the Pichia pastoris strain, PPS-9016 [Pichia pastoris BG16 (pep4Δ, prb1Δ, protease-deficient)] purchased from ATUM was used. According to the ATUM manual, the linearized pPICZFRT-bTRANSFERRIN (N495Q) vector was introduced into Pichia pastoris BG16 by electroporation and transformed. According to the resistance to zeocin (250 ug / mL) antibiotic (Gibco TM , catalog number: R25001), the transformed strains were selected in YPDS agar medium (10 g / L yeast extract, 20 g / L peptone, 182.2 g / L sorbitol, 20 g / L bacto agar). The AOX1 promoter locus integrated into the genome was confirmed by a forward primer (SEQ ID NO: 10) that binds 21 bp in front of the AOX1 promoter in the genome and a reverse primer (SEQ ID NO: 11) that binds to the rhamnose promoter of the vector. The PCR reaction used the Phire Plant Direct PCR kit (Thermo Scientific TM , catalog number: F130WH) and followed the manufacturer's protocol.

[0083] The clones confirmed to have undergone transformation by PCR were inoculated into 1 mL of YP (10 g / L yeast extract, 20 g / L peptone) + 1% rhamnose liquid medium and cultured at 30 °C and 200 rpm for 24 hours. To select the clones in which the FRT region was deleted, since the Flp recombinase (whose expression is induced by rhamnose) recognizes the FRT site, they were streaked on YPD agar medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L bacto agar) and cultured at 30 °C for 72 hours. The single colonies were streaked on YPD agar medium and YPDS + zeocin agar medium respectively, so as to select the colonies in which the FRT region was deleted and thus lost resistance to zeocin. The selected strain was named CF04-1023.

[0084] Example 3. Preparation of Pichia pastoris strains into which PDI orthologs from various strains were introduced and confirmation of bovine transferrin expression

[0085] Example 3-1. Preparation of vectors for introducing PDI from various strains

[0086] To confirm the effect of introducing PDI from various strains into the CF04-1023 strain (the strain expressing bovine transferrin prepared in Example 2) on bovine transferrin expression, vectors introducing PDI from various strains were prepared by the following method.

[0087] As PDI orthologs, based on NCBI BlastP of the PDI amino acid sequence of Pichia pastoris (SEQ ID NO:12), PDI of Hansenula angustata (SEQ ID NO:13) with 54.63% homology to the amino acid sequence of SEQ ID NO:12 and PDI of Trichoderma reesei (SEQ ID NO:14) with 44.20% homology to it were selected as test candidates.

[0088] Specifically, for expression in Pichia pastoris, gene fragments of Pichia pastoris PDI were obtained by PCR using primers with base sequences of SEQ ID NO:15 and SEQ ID NO:16 from gDNA. The sequences of Hansenula angustata PDI and Trichoderma reesei PDI are shown in SEQ ID NO:17 and SEQ ID NO:18. Each sequence was synthesized for cloning by adding 20 bp of complementary sequences before and after the sequence of the pPICZαA vector (Invitrogen TM , catalog number: V19020). Using each synthesized DNA as a template, gene fragments were obtained by PCR using primers with base sequences of SEQ ID NO:19 and SEQ ID NO:20. Using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method, each obtained gene fragment was cloned using the pPICZαA vector linearized with EcoRI-HF (NEB, catalog number: R3101S) to obtain recombinant plasmids. The vector introducing Pichia pastoris PDI was named pPICZA-PDI(PP), the vector introducing Hansenula angustata PDI was named pPICZA-PDI(OA), and the vector introducing Trichoderma reesei PDI was named pPICZA-PDI(TR).

[0089] Example 3-2. Preparation of Pichia pastoris Strains Introducing PDI from Various Strains

[0090] To separately introduce the pPICZA-PDI(PP), pPICZA-PDI(OA), and pPICZA-PDI(TR) vectors prepared in Example 3-1 into the CF04-1023 strain and the Pichia pastoris BG16 strain expressing bovine transferrin prepared in Example 2, strains into which PDI from various strains was introduced were prepared through the following transformation.

[0091] Specifically, the AOX1 promoter site of each vector was linearized using PmeI (NEB, catalog number: R0560S). Subsequently, each linearized vector was purified using the Expin TM PCR SV kit (GeneAll, catalog number: 103-102). According to the manual of ATUM Corporation, each linearized vector was introduced into the CF04-1023 strain by electroporation, and the transformed strains were selected in YPDS agar medium based on the resistance to the zeocin (250 μg / mL) antibiotic. The AOX1 promoter site integrated into the genome was confirmed by a forward primer (SEQ ID NO: 10) bound to 21 bp in the front and a reverse primer (SEQ ID NO: 21) bound to the TEF1 promoter of the vector. The PCR reaction used the Phire Plant Direct PCR kit and followed the manufacturer's protocol. For the selected strains, the strain into which Pichia pastoris PDI was introduced was named CF04-1024, the strain into which Hansenula angustata PDI was introduced was named CF04-1025, and the strain into which Trichoderma reesei PDI was introduced was named CF04-1026.

[0092] Example 3-3. Confirmation of the expression level of bovine transferrin by introducing PDI from various strains into Pichia pastoris strains

[0093] To confirm the expression level of bovine transferrin by introducing PDI from various strains into Pichia pastoris strains, the four strains (CF04-1023, 1024, 1025, 1026) prepared in Example 3-2 were cultured to confirm the expression of bovine transferrin.

[0094] To cultivate the strain, it was cultured in 25 ml of BMMY liquid medium (1% yeast extract, 2% peptone, 13.4 g / l yeast nitrogen base (without amino acids), 100 mM potassium phosphate pH 6.0, 0.004 mg / l biotin, 1% methanol) in a 250 ml baffled conical flask at 30 °C and 200 rpm for 96 hours, and 1% methanol was additionally supplied every 24 hours to induce continuous expression of the protein under the control of the AOX1 promoter. After fermentation was completed, expression was confirmed by analyzing the culture supernatant. The culture supernatant (from which yeast microbial cells were sedimented by centrifugation) was separated by SDS-PAGE (Laemmli, 1970) method using a 4-20% Tris-glycine polyacrylamide gel, and stained using DIRECTBLUE TM Gel Staining Solution (SCGBIOMAX, catalog number: BDS-1000). The concentration comparison of the band corresponding to transferrin on the gel was analyzed using a Calibrated Densitometer (BIO-RAD, GS-900). In addition, to further confirm whether the band with the corresponding size was transferrin, it was confirmed by LC-MS / MS analysis using the gel digestion (Rosenfeld, 1992) method. The SDS-PAGE results of transferrin expression are shown in Figure 3 and the comparison results of transferrin expression levels are shown in Table 1 below.

[0095] Table 1

[0096]

[0097] As a result, as shown in Figure 3 and Table 1, it was confirmed that the strain (CF04-1023) without additional introduction of PDI did not express bovine transferrin, and bovine transferrin was only expressed in the strain with additional introduction of PDI. In addition, compared with the strains into which Hansenula angustata PDI and Pichia pastoris PDI were introduced, it was confirmed that the bovine transferrin expression level of the CF04-1026 strain (which is the strain into which Trichoderma reesei PDI was introduced) was the most excellent, and the expression level of this strain was significantly higher than that of the strain into which Pichia pastoris PDI was introduced by about 1.3 times.

[0098] Example 4. Preparation of Trichoderma reesei PDI-expressing strain

[0099] Since it was confirmed that the bovine transferrin expression level of the Pichia pastoris strain expressing non-glycosylated bovine transferrin into which Trichoderma reesei PDI was introduced was the most excellent in Example 3-3, a Pichia pastoris strain expressing Trichoderma reesei PDI was prepared by the following method.

[0100] Using Pfu-X DNA polymerase, using pPCIZA-PDI(TR) as a template, and using primers with base sequences of SEQ ID NO:22 and SEQ ID NO:23, a DNA fragment consisting of the AOX1 promoter - Trichoderma reesei PDI - AOX1 terminator was obtained by PCR. pPICZFRT was digested with KpnI-HF and NotI-HF, and then two fragments were cloned using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method to obtain a recombinant plasmid, which was named pPICZFRT-PDI(TR) (see Figure 4 ). The pPICZFRT-PDI(TR) vector was introduced into the Pichia pastoris BG16 strain by the same method as described in Example 2, and finally, Trichoderma reesei PDI was integrated into the genome of the Pichia pastoris BG16 strain, and clones that lost resistance to zeocin were selected by excision of the FRT site. The corresponding strain was named the CF04-1035 strain.

[0101] Example 5. Confirmation of Bovine Albumin Expression in Trichoderma reesei PDI-Expressing Strains

[0102] To confirm whether the expression levels of other types of proteins besides bovine transferrin are excellent, the bovine albumin gene was introduced into the Trichoderma reesei PDI-expressing strain (CF04-1035) prepared in Example 4 and the Pichia pastoris BG16 strain, respectively, to compare the expression levels. As the amino acid sequence of bovine albumin, the sequence at positions 25 - 607 except for the signal sequence in Uniprot ID: P02769 was used, and it is shown as SEQ ID NO:24.

[0103] To secrete and express bovine albumin (SEQ ID NO:24), the DNA sequence encoding it was codon-optimized, and for cloning, by using the pPICZαA vector (Invitrogen TM, complementary sequences of 20 bp each were added to the front and back of the sequence (catalog number: V19520) to synthesize it, and this sequence is shown as SEQ ID NO:25. Using Pfu-X DNA polymerase, with the synthesized DNA as a template, and primers with the base sequences of SEQ ID NO:26 and SEQ ID NO:27, a gene fragment was obtained by PCR. In addition, using the pPICZαA vector as a template, and primers with the base sequences of SEQ ID NO:28 and SEQ ID NO:29, a gene fragment used as a vector was obtained by PCR. The two obtained gene fragments were cloned using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method to obtain a recombinant plasmid. The obtained vector was named pPCIZαA-bALBUMIN. By the same method as described in Example 2, the pPCIZαA-bALBUMIN vector was introduced into the BG16 strain and the CF04-1035 strain respectively, and confirmation was carried out. The strain in which the gene encoding bovine albumin was introduced into the Pichia pastoris BG16 strain was named CF04-1005, and the strain in which this gene was introduced into CF04-1035 was named CF04-1038.

[0104] By culturing these two strains, the expression levels of bovine albumin were compared and shown in Figure 5 and Table 2 below.

[0105] Table 2

[0106]

[0107] As a result, as shown in Figure 5 and Table 2, it was confirmed that the expression level of bovine albumin in the CF04-1038 strain (the strain expressing Trichoderma reesei PDI) was approximately 1.4 times higher than that in the CF04-1005 strain (the strain not introduced with Trichoderma reesei PDI).

[0108] Example 6. Confirmation of the expression of bovine insulin precursor in Trichoderma reesei PDI-expressing strains

[0109] To confirm whether the expression of bovine insulin precursor in Trichoderma reesei PDI expression strains is excellent, the gene encoding bovine insulin precursor was introduced into the Trichoderma reesei expression strain (CF04-1035) prepared in Example 4 and the Pichia pastoris BG16 strain respectively, and the expression levels were compared. As the amino acid sequence of bovine insulin precursor, the sequence corresponding to positions 1 to 30 of insulin B chain and the sequence corresponding to positions 61 to 81 of insulin A chain in Uniprot ID: I7CLV3 were used. And as the spacer region, the EPK sequence was added in front, and the AAK sequence was added between the B chain and the A chain (Thomas Kjeldsen, 2002, JBC vol 277, issue 21). This sequence is shown in SEQ ID NO: 25.

[0110] To secrete and express the bovine insulin precursor protein (SEQ ID NO: 30), the DNA sequence encoding it was codon-optimized, and for cloning, the complementary sequence of the pPICZαA vector sequence was synthesized by adding 20 bp at the front and back, and this sequence is shown in SEQ ID NO: 31. Using the synthesized DNA as a template, gene fragments were obtained by PCR using primers with the base sequences of SEQ ID NO: 26 and SEQ ID NO: 27. In addition, using the pPICZαA vector as a template, gene fragments used as vectors were obtained by PCR using primers with the base sequences of SEQ ID NO: 28 and SEQ ID NO: 29. The two obtained gene fragments were cloned using the Gibson assembly (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) method to obtain a recombinant plasmid. The obtained vector was named pPCIZαA-bPROINSULIN. By the same method as described in Example 2, the pPCIZαA-bPROINSULIN vector was introduced into the BG16 strain and the CF04-1035 strain respectively, and confirmation was carried out. The strain in which the gene encoding bovine insulin precursor was introduced into the Pichia pastoris BG16 strain was named CF04-1017, and the strain in which this gene was introduced into CF04-1035 was named CF04-1040.

[0111] The expression levels of bovine insulin precursor were compared by culturing the two strains. Except that 10-20% Tris-tricine polyacrylamide gel was used instead of the 4-20% Tris-glycine polyacrylamide gel in the method described in Example 3-3, the experiment was carried out by the same method, and the results are shown in Figure 6 and Table 3 below.

[0112] Table 3

[0113]

[0114] As a result, as Figure 6 shown in and Table 3, it was confirmed that the expression level of bovine insulin precursor in strain CF04-1040 (a strain expressing Trichoderma reesei PDI) was approximately 1.5 times higher than that in strain CF04-1017 (a strain into which Trichoderma reesei PDI was not introduced).

Claims

1. A method for producing a recombinant protein, comprising: 1) preparing a mutant yeast, including performing the following steps i) and ii) simultaneously, sequentially, or in reverse order: i) introducing a vector containing a gene encoding PDI (protein disulfide isomerase) from a Trichoderma strain into the yeast; ii) introducing a vector containing a gene encoding a target protein into the yeast; and 2) culturing the mutant yeast prepared in step 1).

2. The method for producing a recombinant protein according to claim 1, wherein the Trichoderma strain is Trichoderma reesei.

3. The method for producing a recombinant protein according to claim 1, wherein the PDI (protein disulfide isomerase) from the Trichoderma strain in step 1) i) consists of the amino acid sequence of SEQ ID NO:

14.

4. The method for producing a recombinant protein according to claim 1, wherein the yeast is any one selected from the group consisting of strains of the genera Pichia, Candida, and Hansenula.

5. The method for producing a recombinant protein according to claim 1, wherein the yeast is Pichia pastoris strain.

6. The method for producing a recombinant protein according to claim 1, wherein the target protein in step 1) ii) is a protein containing at least 3 disulfide bonds.

7. The method for producing a recombinant protein according to claim 6, wherein the target protein is at least one selected from the group consisting of transferrin, albumin, and proinsulin.

8. The method for producing a recombinant protein according to claim 7, wherein the transferrin, albumin, and proinsulin are of bovine origin.

9. A mutant yeast for producing a target protein, which contains a gene encoding PDI (protein disulfide isomerase) from a Trichoderma strain.

10. The mutant yeast according to claim 9, wherein the gene encoding PDI from the Trichoderma strain is inserted into the yeast genome.

11. The mutant yeast according to claim 9, wherein the Trichoderma strain is Trichoderma reesei.

12. The mutant yeast according to claim 9, wherein the yeast is any one selected from the group consisting of the genera Pichia, Candida, and Hansenula.

13. The mutant yeast according to claim 9, wherein the mutant yeast is Pichia pastoris strain.

14. The mutant yeast according to claim 9, wherein the target protein contains at least 3 disulfide bonds.

15. The mutant yeast according to claim 9, wherein the target protein is at least one selected from the group consisting of transferrin, albumin, and proinsulin.

16. The mutant yeast according to claim 15, wherein the transferrin, albumin, and proinsulin are of bovine origin.

17. A composition for producing a target protein in a mutant yeast, which contains a gene encoding PDI (protein disulfide isomerase) from a Trichoderma strain or a vector containing the same.

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