Methods, cells, and proteins for improving the expression level of recombinant human albumin.

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 pastoris 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 easy to operate, can be cultured and fermented at a high density on a large scale, and 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, 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 by which two adjacent yeast of different genders mate, and the haploid spores fuse to form a diploid. When the environment is bad, 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 on 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 inhibits the aggregation effect by occupying the binding site of the aggregation protein and preventing it from binding to the mannose residues on the adjacent cell wall.

[0005] The redox state within yeast cells directly affects cell survival, activation, and proliferation. When methanol is used as a carbon source, methanol is broken down into formaldehyde and hydrogen peroxide under the action of alcohol oxidase. However, hydrogen peroxide generates a large amount of reactive oxygen species (ROS) during its metabolic process, directly damaging cellular components such as nucleic acids, lipids, and proteins, and in severe cases causing cell aging or death. Superoxide dimutase can catalyze the superoxide anion radical to produce hydrogen peroxide and oxygen gas. This effectively resists the toxicity of superoxide ions to cells, protects the organism from oxidative damage, maintains the organism's balance, and extends cell lifespan.

[0006] In yeast cells, the endoplasmic reticulum is a crucial site for the synthesis and maturation of secretory proteins and is rich in various molecular chaperones and folderases that assist in protein folding and modification, such as the protein disulfide isomerase PDI. PDI catalyzes the formation and rearrangement of disulfide bonds between cysteine ​​residues, which helps in the precise folding of proteins and can improve their expression levels.

[0007] Human serum albumin (HSA) is a single-chain globular protein consisting of 585 amino acids. When protein processing is inhibited, such as during disulfide bond formation 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 cellular function. There are two main pathways that regulate endoplasmic reticulum stress: the endoplasmic reticulum stress response (UPR) and the endoplasmic reticulum-associated degradation pathway (ERAD). In the UPR, a series of intracellular signaling reactions upregulate the expression levels of folderases and molecular chaperones to improve protein folding ability, while in the ERAD, poorly folded protein substrates are transported from the endoplasmic reticulum to the cytoplasm via a reverse transport mechanism, further marked by ubiquitination, and finally degraded by the proteasome.

[0008] The ubiquitin-proteasome pathway consists of ubiquitin and a series of related enzymes. Ubiquitin-activating enzyme E1 activates ubiquitin molecules in an energy-dependent manner supplied by ATP, and transmits this energy to ubiquitin-conjugating enzyme E2. Ubiquitin ligase E3 recognizes the target protein to be degraded, and ligates the ubiquitin bound to E2 to the Lys residues of the target protein. This process is repeated, and the target protein is bound to multiple ubiquitin molecules. Under the action of the 26S proteasome, the target protein is degraded into oligopeptides consisting of amino acid residues. The proteasome itself is not selective for proteins; the selective specificity lies with the E3 ubiquitin ligase.

[0009] Definition of Terms A "host cell" refers to a cell that accepts exogenous genes during the transformation or transduction process.

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

[0011] A "vector" refers to an autonomous DNA that can introduce exogenous DNA into a host cell, replicate it, or ultimately express exogenous gene DNA. They are mainly divided into cloning vectors, which are primarily used for gene replication and amplification, and expression vectors, which are primarily used for the expression of target genes.

[0012] DNA, or deoxyribonucleic acid, is a type of nucleic acid.

[0013] "Gene expression" refers to the process by which genetic information carried in structural genes within an organism's genome undergoes a series of processes, including transcription and translation, to synthesize specific proteins and subsequently perform their specific biological functions.

[0014] "Manipulable linkage" refers to the covalent linkage of transcriptional and translational regulatory elements to a coding sequence, where the spatial arrangement allows the regulatory elements to guide the expression of the coding sequence.

[0015] A "signal peptide" refers to the N-terminal amino acid sequence that guides the transmembrane transfer of proteins in a newly synthesized polypeptide chain.

[0016] "Molecular chaperones" refer to proteins and polypeptides that help in the precise folding of large molecular structures within cells.

[0017] A "recombinant promoter" refers to a genetically modified or unmodified promoter, 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 located upstream of the 5' end of the target gene coding sequence, recognized and bound to RNA polymerase, and regulates the transcription of the target gene.

[0018] "FDH" Formate dehydrogenase is a type of formate dehydrogenase.

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

[0020] "GAL" Galactose is galactose.

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

[0022] The "UPR" (unfolded protein response) is an endoplasmic reticulum stress response.

[0023] "ERAD" ER-associated degradation is an 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 a slow phenotype that utilizes methanol.

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

[0038] "PCR" stands for Polymerase Chain Reaction.

[0039] "rpm" stands for Rounds per minute, which means rotations per minute.

[0040] "SDS-PAGE" (Sodium dodecyl sulphate polyacrylamide gel electrophoresis) is a type of electrophoresis performed using sodium lauryl sulfate-polyacrylamide gel.

[0041] "SEQ ID NO." (Sequence Identity Document Number) is the sequence number. [Overview of the project] [Problems that the invention aims to solve]

[0042] The object of the invention is to provide a method, cells, and protein for improving the expression level of recombinant human albumin with greater effectiveness, and for specific purposes, refer to several substantial technical effects of specific embodiments. [Means for solving the problem]

[0043] To achieve the above objective, the technical means used in the present invention are as follows. Scheme 1, A method for improving the expression level of recombinant human albumin, characterized by comprising: weakening, disrupting or removing the function of at least one endogenous gene encoding an aggregate protein in recombinant host cells, and / or editing the endogenous gene or a heterologous gene to overexpress superoxide dimutase, and / or editing the endogenous gene or a heterologous gene to overexpress E3 ubiquitin ligase.

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

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

[0046] According to further technical means of the present invention, the yeast cells are one or more species of the yeast genus, which consists of the genera Hansenula, Pichia, and Candida.

[0047] According to further technical means of the present invention, the cells of the genus Pichia are Pichia yeast cells (Pichia pastoris).

[0048] According to further technical means of the present invention, the host cell has had the function of at least one endogenous gene encoding an aggregate protein weakened, disrupted, or removed, wherein the endogenous gene encoding the aggregate protein includes FLO5, FLO8, and FLO11.

[0049] According to further technical means of the present invention, the gene encoding superoxide dimutase is either genetically modified or genetically unmodified.

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

[0051] According to further technical means of the present invention, the gene encoding the E3 ubiquitin ligase is either genetically modified or genetically unmodified.

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

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

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

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

[0056] According to further technical means of the present invention, recombinant promoters include, but are not limited to, AOX1 promoters, GAP promoters, GAL promoters, FDH promoters, and FLD promoters.

[0057] Further technical means of the present invention provide a method for producing recombinant protein, comprising the steps of: weakening, disrupting or removing the function of at least one endogenous gene encoding an aggregate protein in a host cell, wherein the endogenous gene encoding the aggregate protein comprises 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 recombinant protein.

[0058] Further technical means of the present invention provide a method for producing a recombinant protein, comprising the steps of: weakening, disrupting or removing the function of at least one endogenous gene encoding an aggregate protein in a host cell, wherein the endogenous gene encoding the aggregate protein includes FLO5, FLO8 and FLO11; encoding at least one endogenous superoxide dimutase or heterologous superoxide dimutase; encoding human albumin modified with at least one recombinant promoter; and culturing the host cell under conditions suitable for the production of a recombinant protein.

[0059] Further technical means of the present invention provide a method for producing a recombinant protein, comprising the steps of: weakening, disrupting or removing the function of at least one endogenous gene encoding an aggregate protein in a host cell, wherein the endogenous gene encoding the aggregate protein includes FLO5, FLO8 and FLO11; encoding at least one endogenous superoxide dimutase or heterologous superoxide dimutase; encoding at least one endogenous E3 ubiquitin ligase or 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 a recombinant protein.

[0060] A method for improving the expression level of recombinant human albumin, wherein the sequence is SEQ ID NO.1 Saccharomyces budding yeast 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 dimutase coding sequence, SEQ ID NO.5 Human superoxide dimutase coding sequence, SEQ ID NO.6 Yeast E3 ubiquitin ligase code sequence, SEQ ID NO.7 Yeast aggregated protein FLO5 coding sequence, SEQ ID NO.8 Yeast aggregated protein FLO8 coding sequence, SEQ ID NO.9 Yeast aggregated 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 dimutase-F sequence, SEQ ID NO.13 Yeast superoxide dimutase-R sequence, SEQ ID NO.14 Human superoxide dimutase-F sequence, SEQ ID NO.15 Human superoxide dimutase-R sequence, SEQ ID NO.16 Yeast E3 ubiquitin ligase-F sequence, A method characterized by using one or more sequences from among the yeast E3 ubiquitin ligase-R sequences with SEQ ID NO.17.

[0061] Host cells modified with one or more sequence genes from SEQ ID NO.1 to SEQ ID NO.17.

[0062] Recombinant human albumin expressed in host cells modified with one or more sequence genes from SEQ ID NO.1 to SEQ ID NO.17. [Effects of the Invention]

[0063] Compared to the prior art, the present invention using the above-described technical means has the following beneficial effects. This patent can maximize the expression level of recombinant human albumin. The present invention provides a method for improving the expression level of recombinant human albumin by weakening, disrupting or removing the function of at least one endogenous gene encoding an aggregated protein, and / or editing the endogenous gene or a heterologous gene to overexpress superoxide dimutase, and / or editing the endogenous gene or a heterologous gene to overexpress E3 ubiquitin ligase. [Brief explanation of the drawing]

[0064] To further illustrate the present invention, it will be described below with reference to the drawings.

[0065] [Figure 1] This is a schematic diagram of a recombinant expression cassette encoding human albumin. [Figure 2] This is a schematic diagram of a recombinant expression cassette encoding E3 ubiquitin ligase. [Figure 3]This is a schematic diagram of a recombinant expression cassette encoding superoxide dimutase. [Figure 4] This is a schematic diagram of a recombinant expression cassette encoding superoxide dimutase and E3 ubiquitin ligase. [Figure 5] This is a schematic diagram of the recombinant expression cassette for the FLO gene knockout plasmid. [Figure 6] This is a graph showing the SDS-PAGE results for recombinant human albumin expression in Example 3. Lane 1 represents recombinant engineered bacterium IV, lane 2 represents recombinant engineered bacterium V, lane 3 represents recombinant engineered bacterium VI, lane 4 represents recombinant engineered bacterium I, lane 5 represents recombinant engineered bacterium II, and lane 6 represents recombinant engineered bacterium III. The expression level of recombinant human albumin was clearly improved. [Figure 7] This is a graph showing the relative expression levels of recombinant human albumin for each recombinant engineered bacterium in Example 3. [Modes for carrying out the invention]

[0066] This patent provides multiple parallel schemes, where different expressions belong to either improved schemes or parallel schemes based on the basic scheme. Each scheme has its own unique 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 promoters of the yeast cells include, but are not limited to, the AOX1 promoter, GAP promoter, GAL promoter, FDH promoter, and FLD promoter.

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

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

[0072] The yeast cells described above have had the function of at least one endogenous gene encoding an aggregate protein weakened, disrupted, or removed, and include FLO5, FLO8, and FLO11.

[0073] The yeast cells include at least one gene encoding human albumin to which a recombinant promoter is operably ligated, the promoter being preferably a genetically modified or unmodified inducible yeast AOX1 promoter.

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

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

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

[0077] The recombinant promoter is manipulably ligated to a signal peptide sequence, a Saccharomyces budding yeast mating factor signal peptide coding sequence (e.g., as shown in SEQ ID NO. 1), or a human albumin signal peptide coding sequence (e.g., as shown in SEQ ID NO. 2) and a human albumin maturation peptide coding sequence (as shown in SEQ ID NO. 3).

[0078] 3) Expression cassette encoding at least one recombinant nucleic acid molecule that expresses superoxide dimutase The expression cassette includes, but is not limited to, the nucleic acid constructs of 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 ligated to the yeast superoxide dimutase coding sequence. The yeast superoxide dimutase coding sequence is shown in SEQ ID NO. 4.

[0080] The recombinant promoter is operably ligated to the human superoxide dimtase coding sequence. The human superoxide dimtase coding sequence is shown in SEQ ID NO. 5.

[0081] 4) Expression cassette encoding at least one recombinant nucleic acid molecule that expresses E3 ubiquitin ligase The expression cassette includes, but is not limited to, the nucleic acid constructs of 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 ligated to the yeast E3 ubiquitin ligase coding sequence. The yeast E3 ubiquitin ligase coding sequence is shown in SEQ ID NO. 6.

[0083] The present invention involves culturing yeast cells under conditions suitable for the production of recombinant human albumin, inducing the expression of exogenous proteins, and improving the expression level of recombinant human albumin, but is not limited to these methods.

[0084] strain E. coli DH5a competent cells were used in all E. coli cloning experiments.

[0085] Recombinant human albumin host cells are yeast cells, preferably Pichia yeast cells CBS7435, and more preferably GS115 (mutated in the histidinol dehydrogenase His4 gene). + Pichia yeast strain) and KM71 (mutated in the histidinol dehydrogenase His4 gene and disrupted in the AOX1 gene) s This is a Pichia yeast strain.

[0086] Expression vector The expression vector needs to be integrated in single-copy or multi-copy form at a specific site in the host Pichia yeast cell genome and homologous recombination with the chromosome to achieve exogenous gene expression. The pPic9 yeast expression vector (Invitrogen) and the pPicZA yeast expression vector (Invitrogen) are preferred.

[0087] Reagents and culture media These include StuI restriction endonuclease (NEB), PmeI restriction endonuclease (NEB), SacI restriction endonuclease (NEB), Gold High-End Free Maxiplasmid Kit (Kangwei Century), Bleomycin Zeocin (Invitrogen), Geneticin G418 (Gibco), and Ampicillin Sodium (Shanghai Biotechnology Co., Ltd.).

[0088] These are MD medium, LB medium, YPD medium, BMGY medium, and BMMY medium.

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

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

[0091] This is a recombinant superoxide dimutase plasmid (yeast superoxide dimutase or human superoxide dimutase), and its schematic structure is shown in Figure 3.

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

[0093] This is a recombinant expression cassette for the FLO gene knockout plasmid, and its schematic diagram is shown in Figure 5.

[0094] Transformation, screening, and positive clones Plasmids are linearized with DNA restriction endonucleases PmeI, StuI, or SacI, and yeast cells are further transformed according to the electroporation method described in the operating manual for the Pichia yeast expression kit (Invitrogen). The cells are then plated onto selective plates containing the corresponding antibiotic or nutrient requirements and cultured at 30°C for 48 to 72 hours.

[0095] After selecting single colonies grown on plates and culturing them in culture medium, genomic DNA was extracted, and the HSA-F primer sequence (SEQ ID NO. 10) and HSA-R primer sequence (SEQ ID NO. 11) from the sequence listing were used, respectively. PCR is performed using corresponding primers such as yeast superoxide dimutase-F sequence (SEQ ID NO. 12) and yeast superoxide dimutase-R sequence (SEQ ID NO. 13), human superoxide dimutase-F sequence (SEQ ID NO. 14) and human superoxide dimutase-R sequence (SEQ ID NO. 15), and 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, recombinant engineered bacteria containing only the human albumin gene were constructed.

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

[0098] (Example 2) In this example, we constructed an engineered bacterium that enhances the expression level of recombinant human albumin.

[0099] Recombinant engineered bacterium II was prepared by transforming recombinant engineered bacterium I into a recombinant yeast E3 ubiquitin ligase expression cassette (a schematic diagram of this is shown in Figure 2). The yeast E3 ubiquitin ligase coding sequence is shown as SEQ ID NO. 6.

[0100] Recombinant engineered bacteria III were transformed with recombinant human albumin expression cassettes and FLO gene knockout plasmids, respectively. The FLO gene knockout plasmid was constructed, containing homology arm sequences for both sides of the FLO gene to be knocked out, with one selective G418 marker inserted between the two homology arms. A schematic diagram of this plasmid is shown in Figure 5. The coding sequence for yeast agglutinating protein FLO5 is shown in SEQ ID NO. 7, the coding sequence for yeast agglutinating protein FLO8 is shown in SEQ ID NO. 8, and the coding sequence for yeast agglutinating protein FLO11 is shown in SEQ ID NO. 9.

[0101] Recombinant engineered bacteria IV were transformed with recombinant human albumin expression cassettes and recombinant yeast superoxide dimutase expression cassettes, respectively (a schematic diagram of which is shown in Figure 3). The yeast superoxide dimutase coding sequence is shown as SEQ ID NO. 4.

[0102] Recombinant engineered bacteria V was created by transforming recombinant engineered bacteria III as a host cell with recombinant yeast E3 ubiquitin ligase and recombinant yeast superoxide dimutase expression cassettes, and a schematic diagram of this is shown in Figure 4.

[0103] Recombinant engineered bacterium VI was created by transforming recombinant engineered bacterium III as a host cell with recombinant yeast E3 ubiquitin ligase and human superoxide dimutase expression cassettes. A schematic diagram of this structure is shown in Figure 4. The human superoxide dimutase coding sequence is shown in SEQ ID NO. 5.

[0104] (Example 3) As shown in Table 1, recombinant engineered bacteria I, II, III, IV, V, and VI were used, respectively. They were inoculated into 40 ml of BMGY culture medium and cultured overnight at 220 rpm and 30°C. The cell saturation was centrifuged, the precipitate was resuspended in 40 ml of BMMY culture medium, and induction was started at 220 rpm and 25°C. 0.4% methanol was added every 24 hours, and after 96 hours, the mixture was centrifuged at 10000 rpm for 5 minutes, and the supernatant was collected. [Table 1]

[0105] SDS-PAGE electrophoresis was performed on the supernatant (graph shown in Figure 6). When the expression level of recombinant human albumin of recombinant engineered bacterium I was set as the control (100%), it was observed that the expression level of recombinant human albumin of each recombinant engineered bacterium was increased (graph shown in Figure 7). The relative expression level of recombinant human albumin of recombinant engineered bacterium 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 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 be referred to as "human blood albumin" and / or "HSA" and / or "HA" and / or "pdHSA".

[0107] Furthermore, this patent enables the construction of various types of fungal species, and similar types of fungal species are also covered within the scope of this patent.

[0108] Currently, the most common method for mass-producing recombinant human albumin through microbial expression is primarily the yeast expression system. However, yeast cells may aggregate too quickly during their growth and metabolic processes, potentially leading to precipitation from the fermentation broth and incomplete fermentation. If recombinant proteins are inhibited in processes such as disulfide bond formation and transport from the endoplasmic reticulum to the Golgi apparatus, unfolded or misfolded proteins can accumulate in the endoplasmic reticulum, causing endoplasmic reticulum stress and potentially affecting normal cell function. Methanol metabolism generates large amounts of reactive oxygen species, directly damaging cellular components such as nucleic acids, lipids, and proteins, and in severe cases causing cellular senescence or death. The method according to the present invention involves culturing yeast cells under conditions suitable for recombinant human albumin production, inducing protein expression, and improving the expression level of recombinant human albumin, but is not limited to these methods.

[0109] The basic principles, main features, and advantages of the present invention have been described above. Those skilled in the art will understand that the present invention is not limited to the above embodiments, and that the above embodiments and specification are merely for illustrating the principles of the present invention. The present invention may also have various variations and improvements without departing from the spirit and scope of the invention, and all such variations and improvements will be within the scope of the claims.

Claims

1. A method for improving the expression level of recombinant human albumin, The host cells of the above method express recombinant human albumin, and the above method A procedure to weaken, disrupt, or remove the function of at least one endogenous gene encoding an aggregated protein. and the procedure of overexpressing superoxide dismutase by editing endogenous genes or heterologous genes. The method further includes editing an endogenous gene or a heterologous gene to overexpress E3 ubiquitin ligase, E3 ubiquitin ligase is encoded in the yeast E3 ubiquitin ligase coding sequence, SEQ ID NO.

6. The host cell is a Pichia yeast cell (Pichia pastoris), The endogenous genes encoding the aggregated protein include FLO5, FLO8, and FLO11. A method characterized by the following:

2. The endogenous or heterologous gene encoding the superoxide dismutase is either genetically modified or genetically unmodified. A method for improving the expression level of recombinant human albumin according to feature 1.

3. The gene encoding the superoxide dismutase is the yeast superoxide dismutase gene, or the gene encoding the superoxide dismutase is the human copper-zinc superoxide dismutase gene. A method for improving the expression level of recombinant human albumin according to feature 1.

4. The host cell comprises a recombinant expression cassette encoding the human albumin gene, a recombinant expression cassette encoding the superoxide dismutase gene, and a recombinant expression cassette encoding the E3 ubiquitin ligase gene. The expression cassette encoding the human albumin gene, the expression cassette encoding the superoxide dismutase gene, and the recombinant expression cassette encoding the E3 ubiquitin ligase gene are either in one nucleic acid construct or in nucleic acid constructs with different selection markers. A method for improving the expression level of recombinant human albumin according to feature 1.

5. The nucleic acid construct comprises one or more plasmids from among pHIL-D2, pPIC3.5, pHIL-S1, pPIC9, pPink-LC, pPink-HC, pPICZA, pPICZB, pPICZC, pPICZaA, pPICZaB, and pPICZaC, and comprises one or more recombinant promoters from among 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 feature 4.

6. The host cell has human albumin modified with at least one recombinant promoter, the host cell has superoxide dismutase modified with at least one recombinant promoter, and the host cell has E3 ubiquitin ligase modified with at least one recombinant promoter. A method for improving the expression level of recombinant human albumin according to feature 5.

7. Human albumin is the human albumin mature peptide coding sequence with SEQ ID NO.

3. The superoxide dismutase is the yeast superoxide dismutase coding sequence SEQ ID NO. 4 and / or the human superoxide dismutase coding sequence SEQ ID NO.

5. A method for improving the expression level of recombinant human albumin according to feature 1.

Images