Method for improving the expression level of recombinant human albumin, cell and protein

Abstract

The present invention discloses a method for improving the expression level of recombinant human albumin, a cell and a protein, and belongs to the technical field of biopharmaceuticals. In the present invention, while expressing recombinant human albumin, the function of an endogenous gene encoding at least one aggregation protein is weakened, disrupted or removed, and / or the endogenous gene or a heterologous gene is edited to overexpress superoxide dismutase, and / or the endogenous gene or a heterologous gene is edited to overexpress an E3 ubiquitin ligase. Thereby, the expression level of recombinant human albumin is improved.

Description

Technical Field

[0001] The present invention relates to a method for improving the expression level of recombinant human albumin, and particularly to a method for improving the expression level of recombinant human albumin, cells, and proteins.

Background Art

[0002] Pichia yeast is genetically stable, has a strong and strictly regulated promoter, and has an intracellular environment and a glycosylation processing system suitable for the accurate folding of eukaryotic gene products. It is an easy-to-operate eukaryotic expression system for producing proteins that can be cultured and fermented at high density on a large scale and has been widely used in recent years.

[0003] Yeast has two modes of reproduction: asexual reproduction and sexual reproduction. When yeast reproduces asexually, it is stimulated by external environmental pressure, causing yeast aggregation. When the environmental pressure is high, it usually dies. On the other hand, sexual reproduction is the process in which two adjacent yeasts of different genders mate, and haploid spores fuse to form a diploid. In a bad environment, meiosis occurs, and the diploid undergoes meiosis to produce haploid spores.

[0004] Yeast aggregation is an asexual, reversible, and calcium-dependent process, which is a multicellular aggregation phenomenon formed by the binding of the aggregation protein encoded by the aggregation gene and the mannose of the adjacent cell wall. If the cells aggregate too early, they will precipitate from the fermentation broth, which may cause incomplete fermentation. By weakening, disrupting, or removing the function of at least one endogenous gene encoding an aggregation protein, including FLO5, FLO8, and FLO11, the formation of pseudohyphae is inhibited. The addition of mannose to the medium can inhibit the aggregation effect by occupying the binding site of the aggregation protein and preventing it from binding to the mannose residues of the adjacent cell wall.

[0005] The redox state within yeast cells directly affects cell survival, activation, and proliferation. When using methanol as a carbon source, methanol is decomposed into formaldehyde and hydrogen peroxide under the action of alcohol oxidase. Meanwhile, hydrogen peroxide generates a large amount of reactive oxygen species (ROS) during the metabolic process, directly damaging cell components such as nucleic acids, lipids, and proteins, and in severe cases, causing cell aging or death. Superoxide dismutase can catalyze superoxide anions to generate hydrogen peroxide and oxygen gas, effectively resisting the toxicity of superoxide ions to cells, protecting the body from oxidative damage, maintaining the body's balance, and extending cell lifespan.

[0006] In yeast cells, the endoplasmic reticulum is an important site where secretory proteins are synthesized and matured, and is rich in various molecular chaperones, foldases, etc. that assist in protein folding and modification, such as protein disulfide isomerase PDI. PDI can catalyze the formation and rearrangement of disulfide bonds between cysteine residues, help with the accurate folding of proteins, and improve its expression level.

[0007] Human Serum Albumin (HSA) is a single-chain globular protein consisting of 585 amino acids. When protein folding is inhibited during processes such as the formation of disulfide bonds and transport from the endoplasmic reticulum to the Golgi apparatus, unfolded or misfolded proteins accumulate in the endoplasmic reticulum, causing endoplasmic reticulum stress and affecting normal cell function. There are mainly two pathways to regulate endoplasmic reticulum stress, namely the unfolded protein response (UPR) and the endoplasmic reticulum-associated degradation pathway (ERAD). In the UPR, through a series of intracellular signal transduction reactions, the expression levels of foldases and molecular chaperones are upregulated to improve protein folding ability. In ERAD, misfolded protein substrates are transported from the endoplasmic reticulum to the cytoplasm by retrograde transport, further marked by ubiquitination, and finally degraded by the proteasome.

[0008] The ubiquitin-proteasome pathway consists of ubiquitin and a series of related enzymes. Dependent on the energy supplied from ATP, the ubiquitin-activating enzyme E1 activates ubiquitin molecules, transfers them to the ubiquitin-conjugating enzyme E2, and the ubiquitin ligase E3 recognizes the target protein to be degraded, binds to E2, and ligates the ubiquitin to the Lys residue of the target protein. This process is repeated to bind the target protein to multiple ubiquitins. Under the action of the 26S proteasome, the target protein is degraded into oligopeptides consisting of amino acid residues. The proteasome itself has no selectivity for proteins, and it is the E3 ubiquitin ligase that has selectivity.

[0009] Definition of Terms "Host cell" refers to a cell that accepts an exogenous gene during the process of transformation or transduction.

[0010] "Expression cassette" refers to a gene expression system that contains all the necessary elements required for the expression of an exogenous protein, including a promoter, an exogenous gene cloning site, a signal peptide sequence, a mature peptide coding sequence of the target protein, a terminator, a screening marker, etc.

[0011] "Vector" refers to an autonomous DNA that can introduce exogenous DNA into a host cell, replicate it, or finally express the exogenous gene DNA. It is mainly divided into a cloning vector mainly used for gene replication, amplification, etc., and an expression vector mainly used for the expression of a target gene.

[0012] "DNA", Deoxyribonucleic acid, is deoxyribonucleic acid.

[0013] "Gene expression" refers to the process in which the genetic information carried by the structural gene in the biological genome undergoes a series of processes such as transcription and translation to synthesize a specific protein and further exert its specific biological function.

[0014] "Operable linkage" means that a transcriptional regulatory element and a translational regulatory element are covalently linked to a coding sequence, and by virtue of their spatial arrangement, the regulatory element can guide the expression of the coding sequence.

[0015] "Signal peptide" refers to an N-terminal amino acid sequence that guides the transmembrane translocation of a protein in a newly synthesized polypeptide chain.

[0016] "Molecular chaperone" refers to proteins and polypeptides that assist in the correct folding of large molecular structures within a cell.

[0017] "Recombinant promoter" refers to a promoter that is genetically modified or unmodified, a promoter that does not naturally exist upstream of a gene in the genome, or a wild-type promoter. A recombinant promoter is a specific DNA sequence that exists upstream of the 5' end of a target gene coding sequence, is recognized and bound by RNA polymerase, and controls the transcription of the target gene.

[0018] "FDH" Formate dehydrogenase is formate dehydrogenase.

[0019] "FLD" Formaldehyde dehydrogenase is formaldehyde dehydrogenase.

[0020] "GAL" Galactose is galactose.

[0021] "GAP" Glyceraldehyde-3-phosphate dehydrogenase is glyceraldehyde-3-phosphate dehydrogenase.

[0022] "UPR" unfolded protein response is the endoplasmic reticulum stress response.

[0023] "ERAD" ER associated degradation is the endoplasmic reticulum-associated degradation pathway.

[0024] "ROS", Reactive Oxygen Species, is reactive oxygen.

[0025] "HSA", Human Serum Albumin, is human serum albumin.

[0026] "PDI", Protein Disulfide Isomerase, is protein disulfide isomerase.

[0027] "LB", Luria bertani medium, is a medium.

[0028] "MD", Minimal Dextrose medium, is a medium.

[0029] "BMGY", Buffered Glycerol - complex Medium, is a medium.

[0030] "BMMY", Buffered Methanol - complex Mdeium, is a medium.

[0031] "YPD", Yeast extract / peptone / dextrose - media, is a medium.

[0032] "Zeo", zeocin, is bleomycin.

[0033] "His", Histidinol dehydrogenase, is histidinol dehydrogenase.

[0034] "Da", Dalton, is dalton.

[0035] "ml", milliliter, is milliliter.

[0036] "Mut s"Methanol utilization slow" is the methanol utilization slow phenotype.

[0037] "Mut" + "Methanol utilization plus" is the methanol utilization plus phenotype.

[0038] "PCR", Polymerase chain reaction, is the polymerase chain reaction.

[0039] "rpm", Rounds per minute, is revolutions per minute.

[0040] "SDS-PAGE", Sodium dodecyl sulphate polyacrylamide gel electrophoresis, is sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0041] "SEQ ID NO.", Sequence Identity Document Number, is the sequence number.

Summary of the Invention

Problems to be Solved by the Invention

[0042] The object of the invention is to provide a method, cells and proteins for improving the expression level of recombinant human albumin with higher effects. For specific objects, refer to the multiple substantial technical effects in the specific implementation part.

Means for Solving the Problems

[0043] To achieve the above object, the technical means used in the present invention are as follows. Scheme 1 A method for improving the expression level of recombinant human albumin, which comprises weakening, disrupting or removing the function of an endogenous gene encoding at least one agglutinin protein in a recombinant host cell, and / or editing an endogenous gene or a heterologous gene to overexpress superoxide dismutase, and / or editing an endogenous gene or a heterologous gene to overexpress an E3 ubiquitin ligase.

[0044] Scheme 2 A method for improving the expression level of recombinant human albumin, the implementation steps of which include: (1) A host cell encoding at least one recombinant human albumin; (2) A host cell in which the function of at least one endogenous gene encoding an agglutinin protein is weakened, disrupted or removed; (3) A host cell encoding at least one endogenous superoxide dismutase or a heterologous superoxide dismutase; (4) A host cell encoding at least one endogenous E3 ubiquitin ligase or a heterologous E3 ubiquitin ligase.

[0045] According to a further technical means of the present invention, the host cell is a yeast cell.

[0046] According to a further technical means of the present invention, the yeast cell is one or more species selected from the genus of yeast consisting of Hansenula, Pichia and Candida.

[0047] According to a further technical means of the present invention, the cell of the genus Pichia is Pichia pastoris.

[0048] According to a further technical means of the present invention, the host cell has weakened, disrupted or removed the function of at least one endogenous gene encoding an agglutinin protein, and the endogenous genes encoding agglutinin proteins include FLO5, FLO8 and FLO11.

[0049] According to a further technical means of the present invention, the gene encoding the superoxide dismutase is either genetically modified or not genetically modified.

[0050] According to a further technical means of the present invention, the gene encoding the superoxide dismutase is yeast superoxide dismutase, more preferably a human copper-zinc superoxide dismutase nucleic acid molecule.

[0051] According to a further technical means of the present invention, the gene encoding the E3 ubiquitin ligase is either genetically modified or not genetically modified.

[0052] According to a further technical means of the present invention, the gene encoding the E3 ubiquitin ligase is yeast E3 ubiquitin ligase, more preferably an E3 ubiquitin ligase nucleic acid molecule.

[0053] According to a further technical means of the present invention, in the host cell, it encodes human albumin modified with at least one recombinant promoter.

[0054] According to a further technical means of the present invention, the recombinant expression cassette encoding the human albumin gene, the superoxide dismutase and the E3 ubiquitin ligase gene is in one nucleic acid construct or in different nucleic acid constructs of a selection marker.

[0055] According to a further technical means of the present invention, the nucleic acid construct includes, but is not limited to, pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB and pPICZaC plasmids.

[0056] According to a further technical means of the present invention, the recombinant promoter includes, but is not limited to, AOX1 promoter, GAP promoter, GAL promoter, FDH promoter and FLD promoter.

[0057] According to a further technical means of the present invention, there is provided a method for producing a recombinant protein, which comprises the steps of: in a host cell, weakening, disrupting or removing the function of an endogenous gene encoding at least one aggregating protein, wherein the endogenous genes encoding aggregating proteins include FLO5, FLO8 and FLO11; encoding human albumin modified with at least one recombinant promoter; and culturing the host cell under conditions suitable for the production of the recombinant protein.

[0058] According to a further technical means of the present invention, there is provided a method for producing a recombinant protein, which comprises the steps of: in a host cell, weakening, disrupting or removing the function of an endogenous gene encoding at least one aggregating protein, wherein the endogenous genes encoding aggregating proteins include FLO5, FLO8 and FLO11; encoding at least one endogenous superoxide dismutase or a heterologous superoxide dismutase; encoding human albumin modified with at least one recombinant promoter; and culturing the host cell under conditions suitable for the production of the recombinant protein.

[0059] According to a further technical means of the present invention, there is provided a method for producing a recombinant protein, comprising: in a host cell, weakening, disrupting or removing the function of an endogenous gene encoding at least one agglutinin protein, wherein the endogenous genes encoding agglutinin proteins include FLO5, FLO8 and FLO11; encoding at least one endogenous superoxide dismutase or a heterologous superoxide dismutase; encoding at least one endogenous E3 ubiquitin ligase or a heterologous E3 ubiquitin ligase; encoding human albumin modified with at least one recombinant promoter; and culturing the host cell under conditions suitable for the production of the recombinant protein.

[0060] A method for improving the expression level of recombinant human albumin, the sequences being SEQ ID NO.1: Coding sequence of the mating factor signal peptide of Saccharomyces cerevisiae SEQ ID NO.2: Coding sequence of the human albumin signal peptide SEQ ID NO.3: Coding sequence of the mature peptide of human albumin SEQ ID NO.4: Coding sequence of yeast superoxide dismutase SEQ ID NO.5: Coding sequence of human superoxide dismutase SEQ ID NO.6: Coding sequence of yeast E3 ubiquitin ligase SEQ ID NO.7: Coding sequence of yeast agglutinin protein FLO5 SEQ ID NO.8: Coding sequence of yeast agglutinin protein FLO8 SEQ ID NO.9: Coding sequence of yeast agglutinin protein FLO11 SEQ ID NO.10: HSA-F primer sequence SEQ ID NO.11: HSA-R primer sequence SEQ ID NO.12: Yeast superoxide dismutase-F sequence SEQ ID NO.13: Yeast superoxide dismutase-R sequence SEQ ID NO.14 Human Superoxide Dismutase-F sequence, SEQ ID NO.15 Human Superoxide Dismutase-R sequence, SEQ ID NO.16 Yeast E3 Ubiquitin Ligase-F sequence, A method characterized by being any one or more of the sequences of SEQ ID NO.17 Yeast E3 Ubiquitin Ligase-R sequence.

[0061] A host cell engineered with any one or more of the sequence genes of SEQ ID NO.1 to SEQ ID NO.17.

[0062] Recombinant human albumin expressed in a host cell engineered with any one or more of the sequence genes of SEQ ID NO.1 to SEQ ID NO.17.

Advantages of the Invention

[0063] Compared with the prior art, the present invention using the above technical means has the following beneficial effects. This patent can maximize the expression level of recombinant human albumin. The present invention weakens, disrupts or removes the function of an endogenous gene encoding at least one aggregation protein, and / or edits an endogenous gene or a heterologous gene to overexpress superoxide dismutase, and / or edits an endogenous gene or a heterologous gene to overexpress E3 ubiquitin ligase, thereby providing a method for improving the expression level of recombinant human albumin.

Brief Description of the Drawings

[0064] To further illustrate the present invention, the following will further explain with reference to the drawings.

[0065]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Modes for Carrying Out the Invention

[0066] This patent provides a plurality of parallel schemes, and different expression parts belong to improved schemes or parallel schemes based on the basic scheme. Each scheme has its own characteristics.

[0067] The object of the present invention is to provide a method for improving the expression level of recombinant human albumin.

[0068] Therefore, the method for improving the expression level of recombinant human albumin according to the present invention includes, but is not limited to, the following 1) to 4).

[0069] 1) Genetically modified yeast cells The recombinant promoter of the yeast cell includes, but is not limited to, AOX1 promoter, GAP promoter, GAL promoter, FDH promoter and FLD promoter.

[0070] The yeast cells include, but are not limited to, the genera Hansenula, Pichia, Schizosacchromyces, Candida, Schizosaccharomyces, Torulopsis, and Aspergillus. Preferably, they are of the genus Pichia, and more preferably, Pichia pastoris.

[0071] The phenotype of the yeast cells is preferably methanol slow utilization type Mut s such as, but not limited to, KM71 and KM71H, or methanol plus utilization type Mut + such as, but not limited to, GS115, X-33, and CBS7435.

[0072] The yeast cells have a weakened, disrupted, or removed function of an endogenous gene encoding at least one agglutinin protein, including FLO5, FLO8, and FLO11.

[0073] The yeast cells include those in which a recombinant promoter is operably linked to at least one gene encoding human albumin, and the promoter is preferably an inducible yeast AOX1 promoter that is genetically modified or unmodified.

[0074] The yeast cells include at least one expression encoding human superoxide dismutase or yeast superoxide dismutase.

[0075] The yeast cells include at least one expression encoding human E3 ubiquitin ligase or yeast E3 ubiquitin ligase.

[0076] 2) An expression cassette encoding at least one recombinant nucleic acid molecule of human albumin Regarding the expression cassette, the nucleic acid construct includes, but is not limited to, pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB, and pPICZaC plasmids.

[0077] The recombinant promoter is operably linked to a signal peptide sequence, a Saccharomyces cerevisiae mating factor signal peptide coding sequence (such as that shown in SEQ ID NO.1) or a human albumin signal peptide coding sequence (such as that shown in SEQ ID NO.2) and a human albumin mature peptide coding sequence (as shown in SEQ ID NO.3).

[0078] 3) An expression cassette encoding at least one recombinant nucleic acid molecule that expresses superoxide dismutase Regarding the expression cassette, the nucleic acid construct includes, but is not limited to, pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB, and pPICZaC plasmids.

[0079] The recombinant promoter is operably linked to a yeast superoxide dismutase coding sequence. The yeast superoxide dismutase coding sequence is as shown in SEQ ID NO.4.

[0080] The recombinant promoter is operably linked to a human superoxide dismutase coding sequence. The human superoxide dismutase coding sequence is as shown in SEQ ID NO.5.

[0081] 4) An expression cassette encoding at least one recombinant nucleic acid molecule that expresses E3 ubiquitin ligase Regarding the expression cassette, the nucleic acid construct includes, but is not limited to, pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB, and pPICZaC plasmids.

[0082] The recombinant promoter is operably linked to the yeast E3 ubiquitin ligase coding sequence. The yeast E3 ubiquitin ligase coding sequence is as shown in SEQ ID NO.6.

[0083] According to the method of the present invention, yeast cells are cultured under conditions suitable for the production of recombinant human albumin, the expression of exogenous proteins is induced, and the expression level of recombinant human albumin is improved, but not limited thereto.

[0084] Strain All Escherichia coli DH5a competent cells were used for all Escherichia coli cloning experiments.

[0085] The recombinant human albumin host cell is a yeast cell, preferably a Pichia yeast cell CBS7435, more preferably GS115 (Mut of the histidinol dehydrogenase His4 gene mutated) + Pichia yeast strain) and KM71 (Mut of the histidinol dehydrogenase His4 gene mutated and the AOX1 gene disrupted) s Pichia yeast strain).

[0086] Expression vector The expression vector needs to be integrated in the form of a single copy or multiple copies at a specific site of the Pichia yeast cell genome, the host, and homologous recombination with the chromosome is carried out to realize the expression of the exogenous gene. pPic9 yeast expression vector (Invitrogen) and pPicZA yeast expression vector (Invitrogen) are preferred.

[0087] Reagents and media StuI restriction endonuclease (NEB), PmeI restriction endonuclease (NEB), SacI restriction endonuclease (NEB), Gold High End Free Maxi Plasmid Kit (ComWin Biotech), Bleomycin zeocin (Invitrogen), Geneticin G418 (Gibco), Ampicillin sodium Ampicillin (Shanghai Sangon Biotech Co., Ltd.).

[0088] MD medium, LB medium, YPD medium, BMGY medium, BMMY medium.

[0089] Recombinant plasmid It is a recombinant human albumin plasmid, and its schematic diagram is as shown in Figure 1.

[0090] It is a recombinant E3 ubiquitin ligase plasmid (yeast E3 ubiquitin ligase or human E3 ubiquitin ligase), and its schematic diagram is as shown in Figure 2.

[0091] It is a recombinant superoxide dismutase plasmid (yeast superoxide dismutase or human superoxide dismutase), and its schematic diagram is as shown in Figure 3.

[0092] It is a recombinant superoxide dismutase (yeast E3 ubiquitin ligase or human E3 ubiquitin ligase) and E3 ubiquitin ligase (yeast superoxide dismutase or human superoxide dismutase) plasmid expression cassette, and its schematic diagram is as shown in Figure 4.

[0093] It is a recombinant expression cassette of FLO gene knockout plasmid, and its schematic diagram is as shown in Figure 5.

[0094] Transformation, screening and positive clones Linearize the plasmid with DNA restriction endonuclease PmeI, StuI or SacI, and further transform yeast cells according to the electroporation method in the operation manual of the Pichia yeast expression kit (Invitrogen). Apply the cells to a selection plate containing the corresponding antibiotic or auxotrophic requirements, and culture at 30 °C for 48 to 72 hours.

[0095] After selecting a single colony grown on the plate and culturing it in a culture solution, extract genomic DNA, and use the HSA-F primer sequence (SEQ ID NO.10) and HSA-R primer sequence (SEQ ID NO.11) in the sequence listing respectively. Perform PCR using corresponding primers such as yeast superoxide dismutase-F sequence (SEQ ID NO.12) and yeast superoxide dismutase-R sequence (SEQ ID NO.13), human superoxide dismutase-F sequence (SEQ ID NO.14) and human superoxide dismutase-R sequence (SEQ ID NO.15), yeast E3 ubiquitin ligase-F sequence (SEQ ID NO.16) and yeast E3 ubiquitin ligase-R sequence (SEQ ID NO.17) to screen for positive clones.

[0096] (Example 1) In this example, a recombinant engineering bacterium containing only the human albumin gene was constructed.

[0097] For recombinant engineering bacterium I, it is a transformed recombinant human albumin expression cassette, and its schematic configuration diagram is as shown in Figure 1. The signal peptide coding sequence is as shown in SEQ ID NO.1, and the human albumin mature peptide coding sequence is as shown in SEQ ID NO.3.

[0098] (Example 2) In this example, an engineering bacterium for improving the expression level of recombinant human albumin was constructed.

[0099] For recombinant engineering bacterium II, it is obtained by using recombinant engineering bacterium I as the host cell and transforming it with a recombinant yeast E3 ubiquitin ligase expression cassette (its schematic configuration diagram is as shown in Figure 2). The yeast E3 ubiquitin ligase coding sequence is as shown in SEQ ID NO.6.

[0100] For recombinant engineering bacterium III, it was transformed with a recombinant human albumin expression cassette and a FLO gene knockout plasmid respectively. The FLO gene knockout plasmid was constructed to contain homology arm sequences on both sides of the target gene FLO in the plasmid, and a selective G418 marker was inserted between the two homology arms. Its schematic configuration diagram is as shown in Figure 5. The yeast agglutinin protein FLO5 coding sequence is as shown in SEQ ID NO.7, the yeast agglutinin protein FLO8 coding sequence is as shown in SEQ ID NO.8, and the yeast agglutinin protein FLO11 coding sequence is as shown in SEQ ID NO.9.

[0101] For recombinant engineering bacterium IV, it is obtained by transforming a recombinant human albumin expression cassette and a recombinant yeast superoxide dismutase expression cassette respectively (its schematic configuration diagram is as shown in Figure 3). The yeast superoxide dismutase coding sequence is as shown in SEQ ID NO.4.

[0102] For recombinant engineering bacterium V, using recombinant engineering bacterium III as the host cell, it was transformed with a recombinant yeast E3 ubiquitin ligase and a recombinant yeast superoxide dismutase expression cassette. Its schematic configuration diagram is as shown in Figure 4.

[0103] For recombinant engineering bacterium VI, using recombinant engineering bacterium III as the host cell, it was transformed with a recombinant yeast E3 ubiquitin ligase and a human superoxide dismutase expression cassette. Its schematic configuration diagram is as shown in Figure 4. The human superoxide dismutase coding sequence is as shown in SEQ ID NO.5.

[0104] (Example 3) As shown in Table 1, each of the recombinant engineering bacteria I, II, III, IV, V, and VI was used. They were inoculated into 40 ml of BMGY culture medium and cultured overnight at 220 rpm and 30°C. The cell solution was centrifuged, and the precipitate was resuspended in 40 ml of BMMY culture medium and induced at 220 rpm and 25°C. 0.4% methanol was added every 24 hours. After 96 hours, it was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was collected.

Table 1

[0105] SDS-PAGE electrophoresis detection was performed on the supernatant (the graph is shown in Figure 6). Taking the expression level of recombinant human albumin in recombinant engineering bacteria I as the control (100%), it was observed that the expression levels of recombinant human albumin in each recombinant engineering bacteria were all improved (the graph is shown in Figure 7). The relative expression level of recombinant human albumin in recombinant engineering bacteria VI was 216%.

[0106] The present invention relates to a method for improving the expression level of recombinant human albumin. In this specification, the term "recombinant human albumin" may also be referred to as "recombinant human serum albumin" and / or "recombinant human blood albumin" and / or "rHA" and / or "rHSA". The term "human serum albumin" refers to human albumin extracted from human serum, and may also be referred to as "human blood albumin" and / or "HSA" and / or "HA" and / or "pdHSA".

[0107] In addition, this patent can realize the construction of various bacterial species, and similar bacterial species are also within the protection scope of this patent.

[0108] Currently, the most common method for mass-producing recombinant human albumin by microbial expression is mainly the yeast expression system. However, during the growth and metabolism process of yeast cells, they may aggregate too early, precipitate from the fermentation broth, and cause incomplete fermentation. If the recombinant protein is inhibited during processes such as the formation of disulfide bonds and transport from the endoplasmic reticulum to the Golgi apparatus, unfolded or misfolded proteins may accumulate in the endoplasmic reticulum, causing endoplasmic reticulum stress and potentially affecting the normal functions of the cells. Since the metabolism of methanol generates a large amount of reactive oxygen species, it directly damages cell components such as nucleic acids, lipids, and proteins, and in severe cases, it may cause cell senescence or death. According to the method of the present invention, yeast cells are cultured under conditions suitable for the production of recombinant human albumin, protein expression is induced, and the expression level of recombinant human albumin is improved, but it is not limited thereto.

[0109] The basic principle, main features, and advantages of the present invention have been described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and what is described in the above embodiments and the specification is only for explaining the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention also has various changes and improvements, and all of these changes and improvements are within the scope of the claims.

Claims

**Claim 1** A method for improving the expression level of recombinant human albumin, wherein the host cell of the method expresses recombinant human albumin, and the method comprises an operation of weakening, disrupting or removing the function of an endogenous gene encoding at least one aggregating protein, and / or an operation of editing an endogenous gene or a heterologous gene to overexpress superoxide dismutase, and / or an operation of editing an endogenous gene or a heterologous gene to overexpress an E3 ubiquitin ligase, characterized by the above. **Claim 2** The host cell is a yeast cell, characterizing the method for improving the expression level of recombinant human albumin according to claim 1. **Claim 3** The endogenous genes encoding the aggregating proteins include FLO5, FLO8 and FLO11, characterizing the method for improving the expression level of recombinant human albumin according to claim 1. **Claim 4** The endogenous gene or heterologous gene encoding superoxide dismutase is either genetically modified or not genetically modified, and the endogenous gene or heterologous gene encoding the E3 ubiquitin ligase is either genetically modified or not genetically modified, characterizing the method for improving the expression level of recombinant human albumin according to claim 1. **Claim 5** The gene encoding superoxide dismutase is a yeast superoxide dismutase gene, or the gene encoding superoxide dismutase is a human copper-zinc superoxide dismutase nucleic acid molecule gene, characterizing the method for improving the expression level of recombinant human albumin according to claim 1. **Claim 6** The gene encoding the E3 ubiquitin ligase is a yeast E3 ubiquitin ligase, or the gene encoding the E3 ubiquitin ligase is a human E3 ubiquitin ligase nucleic acid molecule, characterizing the method for improving the expression level of recombinant human albumin according to claim 1. **Claim 7** The yeast cell is one or more species of the genus of yeast consisting of Hansenula, Pichia and Candida, characterizing the method for improving the expression level of recombinant human albumin according to claim 2. **Claim 8** The cells of the genus Pichia are Pichia yeast cells, A method for improving the expression level of recombinant human albumin according to claim 7, characterized in that...

9. The recombinant expression cassette encoding the human albumin gene, encoding the superoxide dismutase gene, and encoding the E3 ubiquitin ligase gene is in one nucleic acid construct or in different nucleic acid constructs of selectable markers. A method for improving the expression level of recombinant human albumin according to claim 1, characterized in that...

10. The nucleic acid construct contains any one or more of the plasmids of pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB, and pPICZaC. The recombinant promoter contains any one or more of the AOX1 promoter, GAP promoter, GAL promoter, FDH promoter, and FLD promoter. A method for improving the expression level of recombinant human albumin according to claim 9, characterized in that...

11. Encoding human albumin modified with at least one recombinant promoter in the host cell, encoding superoxide dismutase modified with at least one recombinant promoter in the host cell, and encoding E3 ubiquitin ligase modified with at least one recombinant promoter in the host cell. A method for improving the expression level of recombinant human albumin according to claim 10, characterized in that...

12. The sequences used are SEQ ID NO. 1 Saccharomyces cerevisiae mating factor signal peptide coding sequence SEQ ID NO. 2 Human albumin signal peptide coding sequence SEQ ID NO. 3 Human albumin mature peptide coding sequence SEQ ID NO. 4 Yeast superoxide dismutase coding sequence SEQ ID NO. 5 Human superoxide dismutase coding sequence SEQ ID NO. 6 Yeast E3 ubiquitin ligase coding sequence SEQ ID NO. 7 Yeast agglutinin protein FLO5 coding sequence SEQ ID NO. 8 Yeast agglutinin protein FLO8 coding sequence SEQ ID NO. 9 Yeast agglutinin protein FLO11 coding sequence SEQ ID NO. 10 HSA-F primer sequence SEQ ID NO. 11 HSA-R primer sequence, SEQ ID NO. 12 yeast superoxide dismutase-F sequence, SEQ ID NO. 13 yeast superoxide dismutase-R sequence, SEQ ID NO. 14 human superoxide dismutase-F sequence, SEQ ID NO. 15 human superoxide dismutase-R sequence, SEQ ID NO. 16 yeast E3 ubiquitin ligase-F sequence, SEQ ID NO. 17 yeast E3 ubiquitin ligase-R sequence, which is any one or more of the sequences, A method for improving the expression level of recombinant human albumin according to any one of claims 1 to 11, characterized in that.

13. For recombinant engineering bacterium I, it is a transformed recombinant human albumin expression cassette, and the signal peptide coding sequence is as shown in SEQ ID NO. 1, and the human albumin mature peptide coding sequence is as shown in SEQ ID NO.

3. For recombinant engineering bacterium II, using recombinant engineering bacterium I as the host cell, it is transformed with a recombinant yeast E3 ubiquitin ligase expression cassette, and the yeast E3 ubiquitin ligase coding sequence is as shown in SEQ ID NO.

6. For recombinant engineering bacterium III, it is transformed with a recombinant human albumin expression cassette and an FLO gene knockout plasmid respectively, and the FLO gene knockout plasmid is constructed. The plasmid contains homology arm sequences on both sides of the gene FLO to be knocked out, and one selective G418 marker is inserted between the two homology arms. The yeast agglutinin protein FLO5 coding sequence is as shown in SEQ ID NO. 7, the yeast agglutinin protein FLO8 coding sequence is as shown in SEQ ID NO. 8, and the yeast agglutinin protein FLO11 coding sequence is as shown in SEQ ID NO.

9. For recombinant engineering bacterium IV, it is transformed with a recombinant human albumin expression cassette and a recombinant yeast superoxide dismutase expression cassette respectively, and the yeast superoxide dismutase coding sequence is as shown in SEQ ID NO.

4. For recombinant engineering bacterium V, using recombinant engineering bacterium III as the host cell, it is transformed with a recombinant yeast E3 ubiquitin ligase and a recombinant yeast superoxide dismutase expression cassette. Regarding recombinant engineering bacterium VI, using recombinant engineering bacterium III as the host cell, it was transformed with a recombinant yeast E3 ubiquitin ligase and a human superoxide dismutase expression cassette. The human superoxide dismutase coding sequence is as shown in SEQ ID NO. 5, As shown in Table 1, each of the recombinant engineering bacteria I, II, III, IV, V, and VI was used and inoculated into 40 ml of BMGY culture medium, cultured overnight at 220 rpm and 30 °C, the cell solution was centrifuged, the precipitate was resuspended in 40 ml of BMMY culture medium, induction was started at 220 rpm and 25 °C, 0.4% methanol was added every 24 hours, after 96 hours, centrifuged at 10,000 rpm for 5 minutes, and the supernatant was collected. 【Table 1】 SDS-PAGE electrophoresis detection was performed on the supernatant. When the expression level of recombinant human albumin of recombinant engineering bacterium I was used as a control (100%), it was observed that the expression levels of recombinant human albumin of each recombinant engineering bacterium were all improved, and the relative expression level of recombinant human albumin of recombinant engineering bacterium VI was 216%. A method for improving the expression level of recombinant human albumin according to any one of claims 1 to 12, characterized in that.

14. A host cell engineered with any one or more of the sequences of SEQ ID NO. 1 to SEQ ID NO.

17.

15. Recombinant human albumin expressed in a host cell engineered with any one or more of the sequences of SEQ ID NO. 1 to SEQ ID NO. 17.

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