A method for increasing the expression of recombinant human albumin, cells and proteins

Abstract

The present invention discloses a method, a cell and a protein for increasing the expression of recombinant human albumin, and belongs to the field of biopharmaceutical technology. The present invention reduces, destroys or removes the function of at least one endogenous gene encoding a flocculant protein while expressing recombinant human albumin, and / or edits the endogenous gene or heterologous gene to overexpress superoxide dismutase; and / or edits the endogenous gene or heterologous gene to overexpress E3 ubiquitin ligase. Thus, the expression of recombinant human albumin is increased.

Description

Technical Field

[0001] The present invention relates to a method for increasing the expression level of recombinant human albumin, and in particular to a method, cell and protein for increasing the expression level of recombinant human albumin. Background Art

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

[0003] Yeast has two modes of reproduction: asexual reproduction and sexual reproduction. When yeast reproduces asexually, when it is stimulated by external environmental pressure, it will cause flocculation of yeast, and usually die when the environmental pressure is high. Sexual reproduction is the mating of two adjacent yeasts of different sexes, and the fusion of haploid spores produces diploids. When the environment is harsh, meiosis occurs, and diploid meiosis produces haploid spores.

[0004] Yeast flocculation is an asexual, reversible, calcium-dependent process, which is a multicellular aggregation phenomenon formed by the binding of flocculation proteins encoded by flocculation genes to mannose on the adjacent cell walls. After premature flocculation, cells will precipitate from the fermentation broth, which may lead to incomplete fermentation. Weakening, destroying or removing the function of at least one endogenous gene encoding flocculation proteins, including FLO5, FLO8 and FLO11, inhibits the formation of pseudohyphae. Adding mannose to the culture medium allows it to occupy the binding site of flocculation proteins and cannot bind to mannose residues on the adjacent cell walls, so that flocculation is inhibited.

[0005] The redox state in yeast cells directly affects the survival, activation and proliferation of cells. When methanol is used as a carbon source, methanol is decomposed into formaldehyde and hydrogen peroxide under the action of alcohol oxidase. Hydrogen peroxide will produce a large amount of reactive oxygen species (ROS) during metabolism, which will cause direct damage to cell components such as nucleic acids, lipids, and proteins. In severe cases, it will also cause cell aging or death. Superoxide dismutase can catalyze superoxide anion free radicals to generate hydrogen peroxide and oxygen. It effectively resists the toxicity of superoxide ions to cells, protects the body from oxidative damage, maintains the body's balance, and prolongs cell life.

[0006] In yeast cells, the endoplasmic reticulum is an important site for the synthesis and maturation of secretory proteins. It is rich in various molecular chaperones and folding enzymes 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 proteins fold correctly, and increase their expression levels.

[0007] Human serum albumin (HSA) is a single-chain globular protein composed of 585 amino acids. When the process of disulfide bond formation and protein transport from the endoplasmic reticulum to the Golgi apparatus is blocked, unfolded or misfolded proteins aggregate in the endoplasmic reticulum, causing endoplasmic reticulum pressure, thus affecting the normal function of cells. There are two main pathways to regulate endoplasmic reticulum pressure, namely the unfolded protein response (UPR) and the endoplasmic reticulum-associated degradation pathway (ERAD). UPR uses a series of signal transduction reactions in the cell to increase the expression of folding enzymes and molecular chaperones to improve the folding ability of proteins; ERAD transports incorrectly folded protein substrates from the endoplasmic reticulum to the cytoplasm through reverse transport, and further ubiquitination marks are finally degraded by the proteasome.

[0008] The ubiquitin proteasome pathway consists of ubiquitin and a series of related enzymes. Ubiquitin activating enzyme E1 relies on the energy supplied by ATP to activate ubiquitin molecules and pass them to ubiquitin conjugating enzyme E2. Ubiquitin ligase E3 recognizes the target protein to be degraded and connects the ubiquitin bound to E2 to the Lys residue of the target protein. This process is repeated so that the target protein is bound to multiple ubiquitins. Under the action of 26S proteasome, it is degraded into small peptides composed of amino acid residues. The proteasome itself has no selectivity for proteins, and it is the E3 ubiquitin ligase that has selective specificity.

[0009] Defining Terms

[0010] "Host cell" refers to a cell that receives foreign genes during transformation or transduction.

[0011] "Expression cassette" refers to a gene expression system that contains all the necessary elements required for the expression of foreign proteins, including a promoter, a foreign gene cloning site, a signal peptide sequence, a mature peptide coding sequence of the target protein, a terminator, a selection marker, etc.

[0012] "Vector" refers to autonomous DNA that can bring foreign DNA into host cells for replication or ultimately expression of foreign gene DNA. It is mainly divided into cloning vectors and expression vectors. The former is mainly used for gene replication and amplification, while the latter is mainly used for the expression of target genes.

[0013] "DNA" Deoxyribonucleic acid, deoxyribonucleic acid.

[0014] "Gene expression" refers to the process by which the genetic information carried by structural genes in the genome of an organism is synthesized into specific proteins through a series of processes such as transcription and translation, thereby exerting its specific biological function.

[0015] "Operably linked" refers to the covalent attachment of transcriptional and translational regulatory elements to a coding sequence in a spatial arrangement such that the regulatory elements can direct the expression of the coding sequence.

[0016] "Signal peptide" refers to the N-terminal amino acid sequence in a newly synthesized polypeptide chain that is used to direct the transfer of proteins across the membrane.

[0017] "Molecular chaperones" refer to proteins and peptides that assist in the correct folding of macromolecular structures within cells.

[0018] "Recombinant promoter" refers to a genetically modified or unmodified promoter that does not naturally exist in the genome upstream of a gene or a wild-type promoter. It exists upstream of the 5' end of the target gene coding sequence and is a specific DNA sequence that is recognized and bound by RNA polymerase to control the transcription of the target gene.

[0019] "FDH" Formate dehydrogenase, formate dehydrogenase.

[0020] “FLD”Formaldehyde dehydrogenase, formaldehyde dehydrogenase.

[0021] "GAL"Galactose, galactose.

[0022] "GAP" Glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase.

[0023] “UPR” unfolded protein response.

[0024] “ERAD” ER associated degradation, endoplasmic reticulum associated degradation pathway.

[0025] “ROS” Reactive Oxygen Species, reactive oxygen species.

[0026] "HSA" Human Serum Albumin, human serum albumin.

[0027] "PDI" Protein Disulfide Isomerase, protein disulfide isomerase.

[0028] "LB" Luria bertani medium, culture medium.

[0029] “MD” Minimal Dextrose medium, culture medium.

[0030] "BMGY" Buffered Glycerol-complex Medium, culture medium.

[0031] "BMMY" Buffered Methanol-complex Mdeium, culture medium.

[0032] “YPD” Yeast extract / peptone / dextrose-media, culture medium.

[0033] "Zeo" zeocin, bleomycin.

[0034] "His" Histidinol dehydrogenase, histidine dehydrogenase.

[0035] "Da" Dalton, Dalton.

[0036] “ml”milliliter, milliliter.

[0037] “Mut s ”Methanol utilization slow, methanol utilization slow phenotype.

[0038] “Mut + ”Methanol utilization plus, methanol utilization fast phenotype.

[0039] “PCR”Polymerase chain reaction, polymerase chain reaction.

[0040] “rpm”Rounds per minute, revolutions per minute.

[0041] "SDS-PAGE" Sodium dodecyl sulphate polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0042] “SEQ ID NO.” Sequence Identity Document Number, sequence number. Summary of the invention

[0043] Purpose of the invention: To provide a method, cell and protein for increasing the expression of recombinant human albumin with better effect. For specific purposes, see the multiple substantial technical effects in the specific implementation section.

[0044] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0045] Option 1:

[0046] A method for increasing the expression of recombinant human albumin, characterized in that it comprises weakening, destroying or removing the function of at least one endogenous gene encoding a flocculant protein in a recombinant host cell, and / or editing the endogenous gene or heterologous gene so that superoxide dismutase is overexpressed; and / or editing the endogenous gene or heterologous gene so that E3 ubiquitin ligase is overexpressed.

[0047] Option 2:

[0048] A method for increasing the expression of recombinant human albumin, characterized in that the implementation steps include:

[0049] (1) a host cell encoding at least one recombinant human albumin;

[0050] (2) a host cell in which the function of at least one endogenous gene encoding a flocculant protein is weakened, destroyed or removed;

[0051] (3) a host cell encoding at least one endogenous or heterologous superoxide dismutase;

[0052] (4) A host cell encoding at least one endogenous or heterologous E3 ubiquitin ligase.

[0053] A further technical solution of the present invention is that the host cell is a yeast cell.

[0054] A further technical solution of the present invention is that the yeast is one or more of the following yeast genera: Hansenula, Pichia and Candida.

[0055] A further technical solution of the present invention is that the cells of the genus Pichia are Pichia pastoris cells.

[0056] A further technical solution of the present invention is that the host cell has weakened, destroyed or removed the function of at least one endogenous gene encoding a flocculant protein, and the endogenous genes of the flocculant protein include FLO5, FLO8 and FLO11.

[0057] A further technical solution of the present invention is that the gene encoding superoxide dismutase is genetically modified or not genetically modified.

[0058] A further technical solution of the present invention is that the gene encoding superoxide dismutase is yeast superoxide dismutase, and more preferably is a human copper-zinc superoxide dismutase nucleic acid molecule.

[0059] A further technical solution of the present invention is that the gene encoding the E3 ubiquitin ligase is genetically modified or not genetically modified.

[0060] A further technical solution of the present invention is that the gene encoding the E3 ubiquitin ligase is a yeast E3 ubiquitin ligase, and more preferably a human E3 ubiquitin ligase nucleic acid molecule.

[0061] A further technical solution of the present invention is that the host cell encodes at least one recombinant promoter-modified human albumin.

[0062] A further technical solution of the present invention is that the recombinant expression cassette encoding the human albumin gene, superoxide dismutase and E3 ubiquitin ligase gene is on one nucleic acid construct or on nucleic acid constructs with different selection markers.

[0063] A further technical solution of the present invention is that 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.

[0064] A further technical solution of the present invention is that the recombinant promoter includes but is not limited to AOX1 promoter, GAP promoter, GAL promoter, FDH promoter and FLD promoter.

[0065] A further technical solution of the present invention is to provide a method for producing recombinant proteins, comprising a host cell in which the function of at least one endogenous gene encoding a flocculant protein has been weakened, destroyed or removed, the endogenous genes of the flocculant protein comprising FLO5, FLO8 and FLO11; encoding at least one recombinant promoter modified human albumin; and culturing the host cell under conditions suitable for producing recombinant proteins.

[0066] A further technical solution of the present invention is to provide a method for producing recombinant proteins, comprising a host cell in which the function of at least one endogenous gene encoding a flocculant protein has been weakened, destroyed or removed, the endogenous genes of the flocculant protein include FLO5, FLO8 and FLO11; encoding at least one endogenous or heterologous superoxide dismutase; encoding at least one recombinant promoter-modified human albumin; and culturing the host cell under conditions suitable for producing recombinant proteins.

[0067] A further technical solution of the present invention is to provide a method for producing recombinant proteins, comprising a host cell in which the function of at least one endogenous gene encoding a flocculant protein has been weakened, destroyed or removed, the endogenous genes of the flocculant protein include FLO5, FLO8 and FLO11; encoding at least one endogenous or heterologous superoxide dismutase; encoding at least one endogenous or heterologous E3 ubiquitin ligase; encoding at least one recombinant promoter-modified human albumin; and culturing the host cell under conditions suitable for producing recombinant proteins.

[0068] A method for increasing the expression of recombinant human albumin, characterized in that the sequence is any one or more of the following sequences:

[0069] SEQ ID NO.1 Saccharomyces cerevisiae mating factor signal peptide coding sequence

[0070] SEQ ID NO.2 Human albumin signal peptide coding sequence;

[0071] SEQ ID NO.3 Human albumin mature peptide coding sequence;

[0072] SEQ ID NO.4 yeast superoxide dismutase coding sequence;

[0073] SEQ ID NO.5 Human superoxide dismutase coding sequence;

[0074] SEQ ID NO.6 Yeast E3 ubiquitin ligase coding sequence;

[0075] SEQ ID NO.7 Yeast flocculation protein FL05 coding sequence;

[0076] SEQ ID NO.8 Yeast flocculation protein FL08 coding sequence;

[0077] SEQ ID NO.9 Yeast flocculation protein FL011 coding sequence;

[0078] SEQ ID NO. 10: HSA-F primer sequence;

[0079] SEQ ID NO.11HSA-R primer sequence;

[0080] SEQ ID NO.12 yeast superoxide dismutase-F sequence;

[0081] SEQ ID NO.13 yeast superoxide dismutase-R sequence;

[0082] SEQ ID NO.14 human superoxide dismutase-F sequence;

[0083] SEQ ID NO.15 human superoxide dismutase-R sequence;

[0084] SEQ ID NO.16 Yeast E3 ubiquitin ligase-F sequence;

[0085] SEQ ID NO.17 Yeast E3 ubiquitin ligase-R sequence.

[0086] A host cell after genetic manipulation using any one or more sequences of SEQ ID NO.1-SEQ ID NO.17.

[0087] Recombinant human albumin expressed by host cells after genetic manipulation using any one or more sequences of SEQ ID NO.1-SEQ ID NO.17.

[0088] The present invention adopting the above technical solution has the following beneficial effects compared with the prior art: the present patent can maximize the expression of recombinant human albumin. The present invention provides a method for increasing the expression of recombinant human albumin by constructing a method of weakening, destroying or removing the function of at least one endogenous gene encoding a flocculant protein, 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 E3 ubiquitin ligase. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] In order to further illustrate the present invention, the following is further described in conjunction with the accompanying drawings:

[0090] Figure 1 Schematic diagram of the structure of the recombinant expression cassette encoding human albumin.

[0091] Figure 2 Schematic diagram of the structure of the recombinant expression cassette encoding E3 ubiquitin ligase.

[0092] Figure 3 Schematic diagram of the structure of the recombinant expression cassette encoding superoxide dismutase.

[0093] Figure 4 Schematic diagram of the structure of the recombinant expression cassette encoding superoxide dismutase and E3 ubiquitin ligase.

[0094] Figure 5 Schematic diagram of the structure of the recombinant expression cassette of the FLO gene knockout plasmid.

[0095] Figure 6 The results of SDS-PAGE of recombinant human albumin expression in Example 3 are shown. Lane 1 is recombinant engineered bacteria IV, lane 2 is recombinant engineered bacteria V, lane 3 is recombinant engineered bacteria VI, lane 4 is recombinant engineered bacteria I, lane 5 is recombinant engineered bacteria II, and lane 6 is recombinant engineered bacteria III. The expression of recombinant human albumin is significantly increased.

[0096] Figure 7 This is a graph showing the relative expression levels of recombinant human albumin of each recombinant engineered bacterium in Example 3. DETAILED DESCRIPTION

[0097] This patent provides a variety of parallel solutions, and the different expressions are improved solutions or parallel solutions based on the basic solution. Each solution has its own unique characteristics.

[0098] The present invention aims to provide a method for increasing the expression level of recombinant human albumin.

[0099] Therefore, the present invention relates to a method for increasing the expression of recombinant human albumin, which includes but is not limited to:

[0100] 1) Genetically modified yeast cells

[0101] The yeast cell recombinant promoter includes but is not limited to AOX1 promoter, GAP promoter, GAL promoter, FDH promoter and FLD promoter.

[0102] The yeast cell includes but is not limited to Hansenula, Pichia, Schizosacchromyces, Candida, Schizosaccharomyces, Torulopsis and Aspergillus. Preferably, it is Pichia, and more preferably, it is Pichia pastoris.

[0103] The phenotype of the yeast cell is preferably a slow methanol utilization type Mut s , including but not limited to KM71, KM71H; or methanol fast utilization type Mut + , including but not limited to GS115, X-33, CBS7435.

[0104] The yeast cell has weakened, destroyed or removed the function of at least one endogenous gene encoding flocculant protein, including FLO5, FLO8 and FLO11.

[0105] The yeast cell comprises a recombinant promoter operably linked to at least one gene encoding human albumin, wherein the promoter is preferably a genetically modified or unmodified inducible yeast AOX1 promoter.

[0106] The yeast cell comprises at least one gene encoding human superoxide dismutase or yeast superoxide dismutase.

[0107] The yeast cell comprises at least one gene expressed as a human E3 ubiquitin ligase or a yeast E3 ubiquitin ligase.

[0108] 2) An expression cassette encoding at least one recombinant nucleic acid molecule of human albumin. The expression cassette and nucleic acid construct include but are not limited to pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB and pPICZaC plasmids.

[0109] The recombinant promoter is operably linked to a signal peptide sequence, a Saccharomyces cerevisiae mating factor signal peptide coding sequence (as shown in SEQ ID NO.1) or a human albumin signal peptide coding sequence (as shown in SEQ ID NO.2), and a human albumin mature peptide coding sequence (as shown in SEQ ID NO.3).

[0110] 3) an expression cassette encoding at least one recombinant nucleic acid molecule expressing superoxide dismutase. The expression cassette and nucleic acid construct include but are not limited to pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB and pPICZaC plasmids.

[0111] The recombinant promoter is operably linked to a yeast superoxide dismutase coding sequence, as shown in SEQ ID NO.4.

[0112] The recombinant promoter is operably linked to a human superoxide dismutase coding sequence as shown in SEQ ID NO.5.

[0113] 4) an expression cassette encoding at least one recombinant nucleic acid molecule expressing an E3 ubiquitin ligase. The expression cassette and nucleic acid construct include but are not limited to pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB and pPICZaC plasmids.

[0114] The recombinant promoter is operably linked to a yeast E3 ubiquitin ligase coding sequence, as shown in SEQ ID NO.6.

[0115] According to but not limited to the method provided by the present invention, yeast cells are cultured under conditions suitable for producing recombinant human albumin to induce the expression of exogenous proteins and increase the expression level of recombinant human albumin.

[0116] strain

[0117] E. coli DH5a competent cells were used for all E. coli cloning experiments.

[0118] The host cell for recombinant human albumin is a yeast cell, preferably a Pichia pastoris cell CBS7435, more preferably a GS115 cell (Mut + Pichia pastoris strains) and KM71 (Muts Pichia pastoris strains).

[0119] Expression vector

[0120] The expression vector needs to be integrated into a specific site of the host Pichia cell genome in a single copy or multiple copies, and undergo homologous recombination with the chromosome to achieve the expression of the foreign gene. The pPic9 yeast expression vector (Invitrogen) and the pPicZA yeast expression vector (Invitrogen) are preferred.

[0121] Reagents and media

[0122] StuI restriction endonuclease (NEB), PmeI restriction endonuclease (NEB), SacI restriction endonuclease (NEB), Gold Medal Endotoxin-free Plasmid Extraction Kit (Kangwei Century), Zeocin (Invitrogen), Geneticin G418 (Gibco), Ampicillin Sodium (Shanghai Sangon Biotechnology Co., Ltd.).

[0123] MD medium, LB medium, YPD medium, BMGY medium, BMMY medium.

[0124] Recombinant plasmid

[0125] Recombinant human albumin plasmid, the structure diagram of which is shown in Figure 1 shown.

[0126] Recombinant E3 ubiquitin ligase plasmid (yeast E3 ubiquitin ligase or human E3 ubiquitin ligase), the schematic diagram of its structure is shown in Figure 2 shown.

[0127] Recombinant superoxide dismutase plasmid (yeast superoxide dismutase or human superoxide dismutase), the schematic diagram of its structure is shown in Figure 3 shown.

[0128] The 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 are shown in the schematic diagram. Figure 4 shown.

[0129] The recombinant expression cassette of the FLO gene knockout plasmid is shown in the schematic diagram. Figure 5 shown.

[0130] Transformation and screening of positive clones

[0131] The plasmid was linearized using DNA restriction endonucleases PmeI, StuI or SacI, and yeast cells were transformed by electroporation according to the operation manual of the Pichia pastoris expression kit (Invitrogen). The cells were plated on selection plates containing corresponding antibiotics or nutritional deficiencies and cultured at 30°C for 48 to 72 hours.

[0132] A single colony grown on the plate was picked and cultured in culture medium, and then genomic DNA was extracted. PCR was performed using corresponding primers such as HSA-F primer sequence (SEQ ID NO.10) and HSA-R primer sequence (SEQ ID NO.11) in the sequence list, 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) in the sequence list to screen positive clones.

[0133] Example 1

[0134] This example constructs a recombinant engineered bacterium containing only the human albumin gene.

[0135] Recombinant engineering bacteria I: transformed recombinant human albumin expression cassette, its structural diagram is as follows Figure 1 The signal peptide coding sequence is shown in SEQ ID NO.1, and the human albumin mature peptide coding sequence is shown in SEQ ID NO.3.

[0136] Example 2

[0137] This example constructs an engineering bacterium that increases the expression of recombinant human albumin.

[0138] Recombinant engineering bacteria II: Using recombinant engineering bacteria I as host cells, transform the recombinant yeast E3 ubiquitin ligase expression cassette (its structural schematic diagram is shown in Figure 2 The yeast E3 ubiquitin ligase coding sequence is shown in SEQ ID NO.6.

[0139] Recombinant engineering bacteria III: transform the recombinant human albumin expression cassette and FLO gene knockout plasmid respectively. Construct the FLO gene knockout plasmid, which contains the homology arm sequences on both sides of the gene to be knocked out FLO, and a selective G418 marker is inserted between the two homology arms. The schematic diagram of its structure is shown in the figure Figure 5The coding sequence of yeast flocculation protein FL05 is shown in SEQ ID NO.7, the coding sequence of yeast flocculation protein FL08 is shown in SEQ ID NO.8, and the coding sequence of yeast flocculation protein FL011 is shown in SEQ ID NO.9.

[0140] Recombinant engineering bacteria IV: transform the recombinant human albumin expression cassette and the recombinant yeast superoxide dismutase expression cassette (the schematic diagram of the structure is shown in Figure 3 The yeast superoxide dismutase coding sequence is shown in SEQ ID NO.4.

[0141] Recombinant engineered bacteria V: Recombinant engineered bacteria III is used as a host cell to transform the recombinant yeast E3 ubiquitin ligase and recombinant yeast superoxide dismutase expression cassettes. The schematic diagram of its structure is shown in the figure Figure 4 shown.

[0142] Recombinant engineered bacteria VI: Recombinant engineered bacteria III is used as a host cell to transform the recombinant yeast E3 ubiquitin ligase and human superoxide dismutase expression cassette. The schematic diagram of its structure is shown in the figure Figure 4 The coding sequence of human superoxide dismutase is shown in SEQ ID NO.5.

[0143] Example 3

[0144] Recombinant engineered bacteria I, II, III, IV, V and VI were taken as shown in Table 1. 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. Induction was started at 220 rpm and 25°C. 0.4% methanol was added every 24 hours. After 96 hours, the cells were centrifuged at 10,000 rpm for 5 minutes and the supernatant was collected.

[0145] Table 1

[0146]

[0147] The supernatant was subjected to SDS-PAGE electrophoresis (see the figure for details) Figure 6 ), with the recombinant human albumin expression of recombinant engineering bacteria I as the control (100%), it can be observed that the recombinant human albumin expression of each recombinant engineering bacteria is increased (see the graph Figure 7 ). The relative expression of recombinant human albumin of the recombinant engineering bacteria VI was as high as 216%.

[0148] The present invention relates to a method for increasing the expression of recombinant human albumin. In the present 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".

[0149] It should be noted that this patent can realize the construction of multiple bacterial species, and similar bacterial species are also within the scope of protection of this patent.

[0150] At present, the most commonly used method for expressing human albumin by genetically recombinant microorganisms that can achieve large-scale production is mainly the yeast expression system. However, due to the premature flocculation during the growth and metabolism of yeast cells, it may precipitate from the fermentation broth, which may lead to incomplete fermentation. When the formation of disulfide bonds and the transport of recombinant proteins from the endoplasmic reticulum to the Golgi apparatus are blocked, the unfolded or misfolded proteins accumulate in the endoplasmic reticulum, causing endoplasmic reticulum pressure, which can affect the normal function of cells. In addition, methanol metabolism will produce a large amount of reactive oxygen, which directly damages cell components such as nucleic acids, lipids, and proteins, and in severe cases, it will also cause cell aging or death. According to but not limited to the method provided by the present invention, under conditions suitable for the production of recombinant human albumin, yeast cells are cultured and protein expression is induced, so that the expression amount of recombinant human albumin can be greatly improved.

[0151] The basic principle, main features and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the protection required.

Claims

1. A method for increasing the expression of recombinant human albumin, characterized in that: The host cell of the method, while expressing recombinant human albumin, also comprises the following operations: Attenuating, destroying or removing the function of at least one endogenous gene encoding a flocculant protein; and editing endogenous or heterologous genes to overexpress superoxide dismutase; and editing endogenous or heterologous genes to overexpress E3 ubiquitin ligases; The host cell is a Pichia pastoris cell; The superoxide dismutase is yeast superoxide dismutase or human copper-zinc superoxide dismutase; the yeast superoxide dismutase coding sequence is shown in SEQ ID NO.4; The coding sequence of human copper-zinc superoxide dismutase is shown in SEQ ID NO.5; The E3 ubiquitin ligase is yeast E3 ubiquitin ligase or human E3 ubiquitin ligase; the yeast E3 ubiquitin ligase coding sequence is shown in SEQ ID NO.6; The endogenous genes encoding flocculant proteins include FLO5, FLO8 and FLO11. The coding sequence of yeast flocculant protein FL05 is shown in SEQ ID NO.7; the coding sequence of yeast flocculant protein FL08 is shown in SEQ ID NO.8; and the coding sequence of yeast flocculant protein FL011 is shown in SEQ ID NO.

9.

2. A method for increasing the expression of recombinant human albumin according to claim 1, characterized in that: The recombinant expression cassettes of the gene encoding human albumin, the gene encoding superoxide dismutase and the gene encoding E3 ubiquitin ligase are on one nucleic acid construct or on nucleic acid constructs of different selection markers.

3. A method for increasing the expression of recombinant human albumin according to claim 2, characterized in that: The nucleic acid construct includes any one or more of pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB and pPICZaC plasmids; the recombinant promoter includes any one or more of AOX1 promoter, GAP promoter, GAL promoter, FDH promoter and FLD promoter.

4. A method for increasing the expression of recombinant human albumin according to claim 3, characterized in that: The host cell encodes at least one human albumin modified by a recombinant promoter; the host cell encodes at least one superoxide dismutase modified by a recombinant promoter; and the host cell encodes at least one E3 ubiquitin ligase modified by a recombinant promoter.

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