Recombinant expression vector for preparing highly expressed human serum albumin, novel host strain and use thereof

Abstract

The present invention relates to a recombinant expression vector for preparing highly expressed human serum albumin, a host strain and a use thereof. By means of optimized design of a target gene, a Pichia pastoris engineered strain containing a high-copy-number exogenous gene and expressing the exogenous gene at a high level is constructed; by means of optimization of a pilot-scale fermentation process, the expression level of recombinant human serum albumin from the gene reaches an ultra-high level of 25.82 g / L; and by means of fermentation broth pretreatment and a purification process using three-step column chromatography, the purity of the target protein reaches 99% or more, and the recovery rate reaches 60% or more.

Description

A recombinant expression vector for preparing high expression of human serum albumin, a novel host bacterium and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology for preparing human serum albumin by genetic engineering technology, and particularly relates to a recombinant expression vector for preparing high expression of human serum albumin, a novel host bacterium and application thereof. BACKGROUND

[0002] The development of molecular biology technology provides many methods and means for producing exogenous proteins by using a biological reactor. So far, various exogenous protein expression systems such as Escherichia coli, yeast, insects and mammalian cells have been developed. The Pichia pastoris gene expression system has become one of the most important hosts for expressing exogenous proteins after nearly thirty years of development. For example, the host bacterium GS115 (Cregg, et al (2009). Methods Enzymol. 463, 169-189; US Patent 4,879,231, Phillips Petroleum, 1989). The system has the characteristics of easy high-density fermentation, stable integration of the target gene in the host genome, effective secretion and moderate glycosylation of the expression product, and inexpensive culture medium. The gene expression system uses the high-efficiency and controllable AOX1 promoter, and has efficiently expressed thousands of exogenous proteins such as HBsAg, TNF, EGF, tetanus toxin C fragment, genetically engineered antibodies and the like. It is confirmed that the system is a high-efficiency, practical and simple exogenous gene expression system, which is characterized by improving the expression amount and maintaining the biological activity of the product. Moreover, the system is very suitable for pilot scale-up and large-scale industrial production.

[0003] The general steps for expressing foreign genes in Pichia pastoris include: 1) inserting foreign genes into recombinant expression vectors of Pichia pastoris; 2) treating linearized recombinant plasmids with endonuclease and then transforming Pichia pastoris strains; 3) spreading the transformation liquid on MD plates for the first round of screening of positive recombinants; 4) using YPD plates containing different concentrations of G418 for the second round of screening of positive recombinants; 5) further identifying the integration of foreign genes in the genome of Pichia pastoris; 6) identifying the expression level of foreign genes by small-scale induction expression; and 7) using bioreactors for large-scale fermentation and preparing recombinant proteins from fermentation broth. In order to improve the expression level of foreign genes in Pichia pastoris, it is usually necessary to screen Pichia pastoris transformants containing high copy number of foreign genes, i.e. to increase the gene dosage in host bacteria; and the screening of high copy number transformants is achieved by gradually increasing the G418 resistance, which is a very tedious and random work. Further investigation shows that there are still many variables between high copy Pichia pastoris transformants and high expression of foreign proteins, and the optimal copy number varies with different target genes, and there is no linear relationship between them. In commonly used series of recombinant expression vectors of Pichia pastoris such as pIC9K and pIC3.5K, G418 resistance genes are introduced. After transforming host bacteria, foreign genes and resistance genes are integrated into the chromosome of Pichia pastoris by homologous recombination. By increasing the G418 resistance, the transformants containing multiple copies of target genes can be screened from the transformant population with a small probability.

[0004] In order to screen the positive clone with high copy number, it is necessary to screen with G418 plates with different concentration gradient. The concentration gradient of G418 in the pre-experiment is set as 0, 0.25, 0.5, 0.75, 1.0, 1.5, 1.75, 2.0, 3.0, 4.0 mg / ml. The screening scheme is introduced according to the protocol recommended by Invitrogen Company. The first method is to prepare G418 plates with different concentration gradient, and to print the positive clones on the HIS4 auxotrophic plate one by one to the G418 gradient plate by photocopying to screen the high copy number clone. The feature of this method is that the workload is huge (before photocopying, it needs to be cultured for 3 times continuously to ensure that the concentration of each single transformant is equivalent when photocopying), the screening difficulty is high, the cycle is long, and the number of screened transformants is relatively small, which cannot be used for high-throughput screening. The second method also needs to prepare G418 plates with different concentration gradient, but the difference is that all the positive transformants grown on the HIS4 auxotrophic plate can be washed down with sterile water or liquid medium, and then diluted to an appropriate concentration, and then a certain amount of the diluted liquid is coated on the G418 plate with different concentration gradient. The operation of this method is relatively simple, and more transformants can be screened. However, for different target genes or different Pichia pastoris host strains, it is necessary to do pre-experiment to explore the test of the dilution of the bacterial liquid. The two screening schemes are the common screening methods used by domestic and foreign scientific and technological workers when using Pichia pastoris expression system to express foreign proteins. However, their common shortcomings are that the use amount of G418 is large, the operation is complicated, the workload is large, time-consuming and laborious, and the universality of the screening method for different Pichia pastoris host strains is poor. SUMMARY

[0005] 1. Technical problems solved by the present application

[0006] The purpose of the present application is to solve the problems of low gene expression level, immature fermentation process control technology (due to large fermentation scale, large fermentation tanks of ten tons or dozens of tons are often needed, and there is lack of precedents and tools for process control and detection of large tanks), and immature purification process of recombinant human serum albumin (due to the particularity of recombinant human serum albumin, the purification scale and equipment of the target protein are particularly large, and the purity requirement of the target protein is particularly high, which requires a purity of more than 99.99%), i.e. the purification steps are as many as ten steps, the purification medium load is low, the purification efficiency is low, and the purification recovery rate is low.

[0007] 2. Technical scheme

[0008] In order to achieve the above purpose, the technical scheme provided by the present application is:

[0009] The present application relates to a recombinant expression vector for preparing high expression of human serum albumin, and the nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO: 1.

[0010] Preferably, the recombinant expression vector comprises:

[0011] (1) a 5' regulatory region from Pichia pastoris, which contains a promoter element, and the 5' regulatory region is selected from the alcohol oxidase AOX1 gene, dihydroxyacetone synthase DAS1 gene or histidinol dehydrogenase HIS4 gene from Pichia pastoris, and the 3' end of the regulatory region is connected to the sequence of (2) below;

[0012] (2) an optimized gene encoding human serum albumin, wherein the nucleotide sequence contained therein is shown in SEQ ID NO: 2, and the amino acid sequence contained therein is shown in SEQ ID NO: 3;

[0013] (3) a 3' termination sequence from Pichia pastoris, and the 3' termination sequence is selected from the 3' termination sequence of the AOX1 gene, AOX2 gene or His4 gene from Pichia pastoris.

[0014] Preferably, it further comprises

[0015] at least one marker gene available for screening in E. coli;

[0016] a DNA fragment of an origin of replication that can replicate in an E. coli host bacterium;

[0017] and further comprises at least two marker genes available for screening in yeast.

[0018] Preferably, the recombinant expression vector is any one of pPIC9, pPIC3, pPICZαABC, pPIC3.5K, pHIL-S1, pHIL-D2, pA0804, pA0815, pGAPZαABC, pPIC6αABC, pPIC9K after linearization by restriction endonuclease SacI or BglII enzyme digestion.

[0019] Preferably, the recombinant expression vector is HSA-pPIC9K after linearization by restriction endonuclease SacI or BglII enzyme digestion, which contains at least one copy of the optimized gene encoding human serum albumin.

[0020] Preferably, in the optimized gene encoding human serum albumin, the preferred codon of the alcohol oxidase (AOX1) gene of Pichia pastoris is selected and controlled to account for 90% of the total number of codons of the encoded optimized gene.

[0021] Preferably, in the optimized gene encoding human serum albumin, three restriction enzyme sites of SalI, Hind III and Xba I are inserted in the optimized gene from 5' to 3' direction in sequence, so that the optimized gene is relatively evenly divided into four large fragments.

[0022] Preferably, in the optimized gene encoding human serum albumin, the continuous G-C pairing is reduced, and the A-T pairing preferred by Pichia pastoris is increased, so that the GC content in the optimized gene is adjusted to 45-50%.

[0023] Preferably, in the optimized gene encoding human serum albumin, the expression reading frame further comprises

[0024] The restriction enzyme BamHI site at the 5' end is inserted into the 5' regulatory region (promoter region) of the AOX1 gene, followed by CCAAACGATG 10 deoxyoligonucleotides (containing the Kozak sequence of eukaryotic genes, i.e. AXXATG), followed by the sequence of the yeast a-mating pheromone leader peptide (consisting of 85 amino acids) derived from Saccharomyces cerevisiae, and then the HSA mature gene is inserted between EcoRI and NotI of the multiple cloning site of the pPIC9K recombinant expression vector.

[0025] The coding sequence AAAAGA of two double basic amino acids (-Lys-Arg-) of lysine and arginine is inserted after the EcoRI enzyme site at the 5' end of the target gene, and the specific sequence is SEQ ID NO: 4; and the double stop codon TAATAG is inserted before the NotI enzyme site at the 3' end of the target gene, and the specific sequence is SEQ ID NO: 5.

[0026] A novel host strain for high expression of human serum albumin, characterized in that the novel host strain is Pichia pastoris HSA-C16, which is preserved in the China General Microbiological Culture Collection Center on March 28, 2024, and the preservation number is CGMCC No. 30175.

[0027] Preferably, the novel host strain is an engineering strain of an expression gene recombinant HSA obtained by introducing the above-mentioned recombinant expression vector for preparing high expression of human serum albumin into Pichia pastoris CBS7435 strain.

[0028] Application of a novel host strain for high expression of human serum albumin, the application of the novel host strain in the preparation of human serum albumin 3. Beneficial effects

[0029] Compared with the prior art, the technical scheme provided by the present application has the following beneficial effects:

[0030] The present application relates to a recombinant expression vector for preparing high expression of human serum albumin, a novel host and its application, through the optimization design of the target gene, the construction of the Pichia pastoris engineering bacteria containing high copy number of exogenous genes and high expression level, and the optimization of the pilot fermentation process, the expression level of the recombinant human serum albumin reaches a super high level of 25.82g / L; the purification process of the recombinant human serum albumin only uses the pretreatment of the fermentation broth and three-step column chromatography, and the purity of the target protein reaches more than 99%, and the recovery rate of the whole purification process reaches more than 60%.

[0031] The biological preservation instruction of Pichia pastoris HSA-C16 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 28, 2024, and the preservation address is No. 1, Beichen West Road, Hua Yuan 3, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No. 30175. BRIEF DESCRIPTION OF DRAWINGS

[0032] Fig. 1 is a computer simulation alignment curve schematic diagram of adjusting the G-C pairing content inside the nucleotide sequence of the optimized gene of the embodiment to the optimal ratio as shown in SEQ ID NO: 2;

[0033] Fig. 2 is a Pichia pastoris multi-copy recombinant expression vector pPIC9K map of the embodiment;

[0034] Fig. 3 is an agarose gel electrophoresis (1% agarose gel) of the optimized gene of the embodiment;

[0035] Fig. 4 is a schematic diagram of the construction process of the recombinant plasmid of the embodiment;

[0036] Fig. 5 is a nucleotide sequence analysis map one of the optimized gene of the embodiment;

[0037] Fig. 6 is a nucleotide sequence analysis map two of the optimized gene of the embodiment;

[0038] Fig. 7 is a nucleotide sequence analysis map three of the optimized gene of the embodiment;

[0039] Fig. 8 is a schematic diagram of the cell state change of the Pichia pastoris host bacteria by electroporation of the recombinant plasmid of the embodiment;

[0040] Fig. 9 is a schematic diagram of the cell state change of the Pichia pastoris GS115 host bacteria by electroporation of the recombinant plasmid of the embodiment;

[0041] Fig. 10 is a schematic diagram of the cell state change of the Pichia pastoris CBS7435 host bacteria by electroporation of the recombinant plasmid of the embodiment;

[0042] Figure 11 is a graph of the results of the small-scale induction experiment of the engineered Pichia GS115 strain prepared in the examples;

[0043] Figure 12 is a graph of the results of the small-scale induction experiment of the engineered Pichia GS115 strain prepared in the examples;

[0044] Figure 13 is a graph of the results of the small-scale induction experiment of the engineered Pichia CBS7435 strain prepared in the examples;

[0045] Figure 14 is a graph of the results of the small-scale induction experiment of the engineered Pichia CBS7435 strain prepared in the examples;

[0046] Figure 15 is a schematic diagram of the protein electrophoretogram of the engineered strain prepared in the examples at different time nodes of the 50L fermenter fermentation test;

[0047] Figure 16 is a schematic diagram of the protein electrophoretogram of the engineered strain prepared in the examples at different time nodes of the 50L fermenter fermentation test;

[0048] Figure 17 is a schematic diagram of the protein electrophoretogram of the engineered strain prepared in the examples at different time nodes of the 50L fermenter fermentation test;

[0049] Figure 18 is a standard curve for the quantitative determination of the protein concentration measured using bovine serum albumin as a standard;

[0050] Figure 19 is a schematic diagram of the separation and purification results of the supernatant sample of the engineered strain prepared in the examples in a 50L fermenter;

[0051] Figure 20 is a purity identification data graph of the serum albumin sample obtained by purification in the examples;

[0052] Figure 21 is a schematic diagram of the non-reduced molecular weight TIC spectrum analysis comparison of the serum albumin sample obtained by purification in the examples and the rHSA national standard;

[0053] Figure 22 is a schematic diagram of the reduced molecular weight TIC spectrum analysis comparison of the serum albumin sample obtained by purification in the examples and the rHSA national standard;

[0054] Figure 23 is a schematic diagram of the reduced molecular weight mass spectrum analysis comparison of the serum albumin sample obtained by purification in the examples and the rHSA national standard. DETAILED DESCRIPTION

[0055] In order to make the personnel in the technical field better understand the scheme of the present application, the technical scheme in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0056] It should be noted that the terms "first", "second", and the like in the specification and claims of the present application and the above-described drawings are used to distinguish similar objects, and do not necessarily have to be used to describe a specific order or sequence. It should be understood that the data thus used can be interchanged under appropriate circumstances, so that the embodiments of the present application described herein can be implemented. In addition, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device including a series of steps or units does not have to be limited to only those steps or units clearly listed, but can include other steps or units not clearly listed or inherent to these processes, methods, products or devices.

[0057] In the present application, the terms "upper", "lower", "left", "right", "front", "back", "top", "bottom", "inner", "outer", "middle", "vertical", "horizontal", "lateral", "longitudinal", and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings. These terms are mainly used to better describe the present application and its embodiments, and are not used to limit the indicated devices, elements or components to have a specific orientation, or to be constructed and operated in a specific orientation.

[0058] In addition, in addition to being used to indicate the orientation or positional relationship, the above-mentioned part of the terms can also be used to indicate other meanings, for example, the term "upper" can also be used to indicate a certain dependent relationship or connection relationship in some cases. For those skilled in the art, the specific meaning of these terms in the present application can be understood according to the specific circumstances.

[0059] In addition, the terms "mount", "set", "provided with", "connected", "connected", "sleeved" should be broadly understood. For example, it can be a fixed connection, a detachable connection, or an integral structure; it can be a mechanical connection, or an electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, or internal communication between two devices, elements or components. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0060] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other in the case of no conflict. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0061] Embodiment 1

[0062] The recombinant expression vector for preparing high expression of human serum albumin in the present embodiment has a nucleotide sequence as shown in SEQ ID NO: 1.

[0063] The recombinant expression vector comprises:

[0064] (1) a 5' regulatory region from Pichia pastoris, which contains a promoter element, and the 5' regulatory region is selected from the group consisting of alcohol oxidase AOX1 gene, dihydroxyacetone synthase DAS1 gene or histidinol dehydrogenase HIS4 gene from Pichia pastoris, and the 3' end of the regulatory region is connected to the sequence of (2) below;

[0065] (2) an optimized gene encoding human serum albumin, wherein the nucleotide sequence contained therein is as shown in SEQ ID NO: 2, and the amino acid sequence contained therein is as shown in SEQ ID NO: 3;

[0066] (3) a 3' termination sequence from Pichia pastoris, and the 3' termination sequence is selected from the group consisting of 3' termination sequence of AOX1 gene, AOX2 gene or His4 gene from Pichia pastoris.

[0067] Further comprising

[0068] at least one marker gene available for screening in E. coli;

[0069] a DNA fragment of an origin of replication available for replication in an E. coli host;

[0070] Further comprising at least two marker genes available for screening in yeast.

[0071] The recombinant expression vector is any one of pPIC9, pPIC3, pPICZαABC, pPIC3.5K, pHIL-S1, pHIL-D2, pA0804, pA0815, pGAPZαABC, pPIC6αABC, pPIC9K after linearization by restriction endonuclease SacI or BglII.

[0072] The recombinant expression vector is HSA-pPIC9K after linearization by restriction endonuclease SacI or BglII, wherein at least one copy of the optimized gene encoding human serum albumin is contained.

[0073] The optimized gene encoding human serum albumin uses the preferred codon of the alcohol oxidase (AOX1) gene of Pichia pastoris and controls the proportion of the preferred codon in the total number of codons in the optimized gene to be 90%.

[0074] The optimized gene encoding human serum albumin has three restriction enzyme sites of SalI, Hind III and Xba I inserted in the optimized gene from 5' to 3' direction, so that the optimized gene is relatively evenly divided into four large fragments.

[0075] The optimized gene encoding human serum albumin reduces the continuous G-C pairing and increases the A-T pairing preferred by Pichia pastoris, so that the GC content in the optimized gene is adjusted to 45-50%.

[0076] The optimized gene encoding human serum albumin also includes an expression reading frame of

[0077] The restriction enzyme BamHI site at the 5' end is inserted into the 5' regulatory region (promoter region) of the AOX1 gene, followed by 10 deoxyoligonucleotides (containing the Kozak sequence of eukaryotic genes, i.e. AXXATG), followed by the sequence of the yeast a-mating pheromone leader peptide (consisting of 85 amino acids) derived from Saccharomyces cerevisiae, and then the HSA mature gene is inserted between EcoRI and NotI in the multiple cloning site of the pPIC9K recombinant expression vector.

[0078] The coding sequence AAAAGA of two double basic amino acids (-Lys-Arg-) is inserted after the EcoRI enzyme site at the 5' end of the target gene, and the specific sequence is SEQ ID NO: 4; the double stop codon TAATAG is inserted before the NotI enzyme site at the 3' end of the target gene, and the specific sequence is SEQ ID NO: 5.

[0079] Example 2

[0080] The new host strain for high expression of human serum albumin in this embodiment is Pichia pastoris HSA-C16, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 30175, and the preservation date is March 28, 2024.

[0081] The new host strain is an engineering strain of an expression gene recombinant HSA obtained by introducing the recombinant expression vector for preparing high expression of human serum albumin as described in Example 1 into Pichia pastoris CBS7435 strain.

[0082] Use of the new host strain in preparing human serum albumin

[0083] The related preparation process and experimental data of the recombinant expression vector of Example 1 and the new host strain of Example 2 are as follows:

[0084] The materials and equipment used include:

[0085] I. Strains

[0086] 1. Pichia pastoris host strain GS115 (Pichia pastoris (his4) NRRL Y-15851);

[0087] 2. Pichia pastoris host strain CBS7435 (Pichia pastoris NRRL Y-11430);

[0088] 3. E. coli host strain:

[0089] E. coli JM109 F' [(recAl, supE44 endAl hsdR17 gyrA96 relAl thi△(lac-proAB)

[0090] F' [traD36 proAB* lacIa lacZ△M15]);

[0091] E. coli HB101 (supE44 hsd S20(rB-mB-) recA: ara-14 proA2 lac Y: galK2 rpsL20 xyl-5 mtl-1).

[0092] The above E. coli host strains are used for cloning of target genes and construction and preparation of plasmids.

[0093] II. Main reagents

[0094] 1. DNA restriction enzymes, T4 DNA ligase, polymerase, etc. are purchased from GIBCO-BRL, Pharmacia, Bio-Labs and Huamei Biological Engineering Co., Ltd. respectively;

[0095] 2. Casein hydrolysate (product of MERK Company, Germany);

[0096] 3. Bacto-yeast extract (product of Difco Company, USA);

[0097] 4. PCR amplification kit (product of Pharmacia Company, Sweden);

[0098] 5. DNA sequence analysis kit (product of USB Company, USA);

[0099] 6. Acrylamide (Acr), N, N-dimethyl bisacrylamide (Bis), Sodium dodecyl sulfate (SDS), Guanidine hydrochloride, Urea, TEMED (Sigma product, UK);

[0100] 7. IPTG, X-gal, DTT, Agarose (Sigma product, UK);

[0101] 8. YNB, Biotin, Agar (Difco product, USA);

[0102] 9. Sorbitol, Glucose, L-histidine, L-lysine, L-methionine, L-leucine, L-isoleucine, L-glutamic acid (Sigma product, USA);

[0103] 10. Glycerol, Methanol (Shanghai Chemical Reagent Factory);

[0104] 11. Enzyme reaction solution:

[0105] Restriction enzyme high salt buffer: 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2;

[0106] Restriction enzyme medium salt buffer: 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl2;

[0107] Restriction enzyme low salt buffer: 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2;

[0108] T4 DNA ligase buffer: 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 10 mM DTT, 1 mM ATP;

[0109] 13. SDS-PAGE protein electrophoresis reagent:

[0110] Electrophoresis buffer: 192 mM glycine, 25 mM Tri-HCl, 0.1% SDS, pH 8.3;

[0111] Concentrated gel buffer: 125 mM Tris-HCl, 0.1% SDS, pH 6.8;

[0112] Separation gel buffer: 375 mM Tris-HCl, 0.1% SDS, pH 8.8;

[0113] Sample buffer (1X): 50 mM Tris-HCl (pH 6.8), 1% SDS, 10% glycerol, 2.5% mercaptoethanol, 0.05% bromophenol blue;

[0114] 30% Acrylamide: 29% acrylamide, 1% N, N-dimethylacrylamide;

[0115] Coomassie brilliant blue staining solution: 0.25% (W / V) Coomassie brilliant blue G-250, 5% Hac;

[0116] 45% Ethanol decolorizing solution: 7.5% Hac, 10% ethanol;

[0117] 13. Commonly used buffers:

[0118] TE buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA;

[0119] STE buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 20 mM NaCl PBS buffer: 10 mM NaH, P0q-Na, HPO3 (pH 7.0), 150 mM NaCl;

[0120] 10X TBS buffer: 108 g Tris base, 55 g boric acid, 40 ml EDTA (0.5 M) per liter, pH 8.0;

[0121] 50X TAE buffer: 242 g Tris base, 57.1 ml glacial acetic acid, 100 ml EDTA (0.5 M) per liter, pH 8.0;

[0122] Saturated phenol: After being distilled, it is saturated with TE buffer and stored at 20°C in a 20 mL aliquot;

[0123] Phenol: chloroform: isoamyl alcohol (V / V): 1:1:0.8;

[0124] Chloroform: isoamyl alcohol (V / V): 24:1.

[0125] III. Main instruments and equipment

[0126] 1. ABI 381A DNA automatic synthesizer, product of American Applied Biosystems (ABI) Company;

[0127] 2. 5 liter RIBE-5 full-automatic fermentation tank, product of Shanghai Guoqiang Biochemical Engineering Equipment Co., Ltd.;

[0128] 3. 15 liter Biocenter 15F full-automatic fermentation tank, product of Shanghai Guoqiang Biochemical Engineering Equipment Co., Ltd. (software system: Fermentation Star);

[0129] 4.50L Biocenter 50F Fully Automatic Fermenter, product of Shanghai Guoqiang Biochemical Engineering Equipment Co., Ltd. (Software system: Fermentation Star);

[0130] 5. 100L Biocenter 100F Fully Automatic Fermenter, product of Shanghai Guoqiang Biochemical Engineering Equipment Co., Ltd. (Software system: Fermentation Star);

[0131] 6,500-liter fully automatic Bplus bioreactor, a product of B. Braun GmbH, Germany;

[0132] 7. SDL-100 protein purification system, Suzhou Saipu Instrument Co., Ltd. (medium-pressure chromatography workstation, SCG software);

[0133] 8. Agilent High Performance Liquid Spectrometer, Agilent Technologies, 1260 Infinity, a product of Agilent Technologies, USA;

[0134] 9. External pressure ultrafiltration membrane module (filtration accuracy: 30KD), product of Huzhou Kelu Membrane Technology Co., Ltd.

[0135] 10. External pressure ultrafiltration membrane module (filtration accuracy: 100KD), product of Huzhou Kelu Membrane Technology Co., Ltd.

[0136] 11. Full-wavelength microplate reader, Thermo Fisher 1510, a product of Thermo Fisher Scientific, USA;

[0137] 12. Electroporation converter, Micropulser 411BR10654, BIO-RAD Corporation, USA.

[0138] IV. Experimental Methods

[0139] (I) Culture medium and culture conditions

[0140] *E. coli* culture medium LB (1% bacto-typtone, 0.5% bacto-yeast extract, 1% NaCl), solid medium with 15 g / L Agar powder, *E. coli* cultured at 37℃; *Pichia pastoris* rich medium YPD (1% Yeast extract, 2% Peptone, 2% Dextrose); *Pichia pastoris* protoplast regeneration medium RBD [1M sorbital, 1% Dextrose, 1.34% YNB, 4×10⁻⁶ ppm] 5Biotin, 0.005% Amino acid mix (including L-glutamic acid, L-methionine, L-leucine, L-isoleucine, L-lysine), 2% Agar powder in liquid media if solid media; Pichia shaker flask media BMGY and BMMY (1% yeast extract, 2% peptone, 100 mM PBS buffer, pH 6.0, 1.34% YNB, 4X105% Biotin, 1% glycerol or 0.5% methanol). Pichia was grown at 30°C.

[0141] (II) Pichia pastoris fermenter high-density fermentation medium and culture conditions: A, 10x Basal Salts: 1. H3PO4, 85% 42ml 2. CaSO2-2H2O 1.8g / L 3. K2SO4 28.6g / L 4. MgSO4-7H2O 23.4g / L 5. KOH 6.5g / L B. 250x PTMl salts: 1. CuSO4-5H2O 6g / L 2. KI 0.08g / L 3. MnSO4-H2O 3g / L 4. Na2MoO4-2H2O 0.2g / L 5. H3BO3 0.02g / L 6. CoCl2 0.5g / L 7. ZnCl2 20g / L 8. FeSO4-7H2O 65g / L 9. Biotin 0.2g / L 10. H2SO4 5ml C. glycerol 8% (V / V) D. Feed medium 50% glycerol (1L) + 12ml / L PTMl E. Induced medium 100% methanol (1L) + 12ml / L PTMl F. Dissolved O2(DO) >20% G. pH 5.0-5.8 H. Temperature 20-30°C

[0142] (III) Pichia pastoris fermenter high-density fermentation medium and culture conditions: A, 10x Basal Salts: 1. H3PO4, 85% 42ml 2. CaSO2-2H2O 1.8g / L 3. K2SO4 28.6g / L 4. MgSO4-7H2O 23.4g / L 5. KOH 6.5g / L B. 250x PTMl salts: 1. CuSO4-5H2O 6g / L 2. KI 0.08g / L 3. MnSO4-H2O 3g / L 4. Na2MoO4-2H2O 0.2g / L 5. H3BO3 0.02g / L 6. CoCl2 0.5g / L 7. ZnCl2 20g / L 8. FeSO4-7H2O 65g / L 9. Biotin 0.2g / L 10. H2SO4 5ml C. glycerol 8% (V / V) D. Feed medium 50% glycerol (1L) + 12ml / L PTMl E. Induced medium 100% methanol (1L) + 12ml / L PTMl F. Dissolved O2(DO) >20% G. pH 5.0-5.8 H. Temperature 20-30°C

[0143] The above YPD medium is used for the cultivation of Pichia pastoris primary seed bacteria.

[0144] Preparation of B liquid: BSM medium (1 L): 1. 98% glycerol 63 g / L 2. ddH2O (ultra-pure water) 912 ml 3. CaSO2·2H2O 0.46 g / L 4. MgSO4·7H2O 5.84 g / L 5. K2SO4 7.34 g / L 6. (NH4)2SO4 9 g / L

[0145] The above BSM medium is used for the cultivation of Pichia pastoris secondary seed bacteria and as the main nutrient medium for the cultivation of yeast in a bioreactor.

[0146] Preparation of C liquid:

[0147] Preparation of sodium hexametaphosphate (HMP) solution (150 ml): 1. (NaPO3)6(Sodium hexametaphosphate) 30 g 2. ddH2O (ultra-pure water) 150 ml

[0148] Preparation of D liquid:

[0149] PTM1 solution (1 L): 1. CuSO4·5H2O 6 g / L 2. KI 0.08 g / L 3. MnSO4·H2O 3 g / L 4. Na2MoO4·2H2O 0.2 g / L 5. H3BO3 0.02 g / L 6. CoCl2 0.5 g / L 7. ZnCl2 20 g / L 8. FeSO4·7H2O 65 g / L 9. Biotin 0.2 g / L 10. H2SO4 5 ml

[0150] (iv) Extraction of plasmid

[0151] 1. Small-scale plasmid extraction

[0152] The single clone was inoculated into 2 ml LB medium containing the corresponding antibiotic and shaken overnight at 37°C. Next day, 1.5 ml of the bacterial solution was taken into an Eppendorf tube, centrifuged to precipitate the bacterial cells, and the supernatant was removed. The bacterial precipitate was placed in an ice bath, and 100 μl of solution I (50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0) was added, followed by shaking and mixing, incubation at room temperature for 5 min, addition of 200 μl of solution II (0.2 N NaOH, 1% SDS), mixing by inversion, ice bath for 5 min, addition of 150 μl of solution III (3 M NaAc, pH 4.8), gentle shaking and mixing, ice bath for 5 min, addition of 450 μl of re-distilled phenol / chloroform (1:1), mixing, centrifugation at 12,000 rpm for 10 min, careful aspiration of the upper aqueous phase into 2 volumes of pre-cooled absolute ethanol, placement in a -20°C refrigerator for 2 hours, centrifugation at 12,000 rpm for 15 min, aspiration of the ethanol, careful addition of 500 μl of 70% ethanol to remove salts, centrifugation to dryness, addition of 18 μl of TE and 2 μl of RNAase enzyme solution, incubation at 37°C for 1 hour, and use for enzyme digestion and further cloning.

[0153] 2. Large-scale plasmid extraction

[0154] 500 ml of the bacterial solution which had been incubated overnight was centrifuged at 5000 rpm for 10 min at 4°C, 100 ml of STE was added, the solution was resuspended and the bacterial cells were collected by centrifugation, 18 ml of solution I (50 mM glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0) and 2 ml of lysozyme (10 mg / ml, 1% SDS) were added, and incubation was carried out at room temperature for 10 min; 40 ml of solution II (0.2 N NaOH, 1% SDS) was added, the solution was mixed gently (until the bacterial solution was transparent), and ice bath was carried out for 5 min; 60 ml of solution III (3 M NaAc, pH 4.8) was added, the solution was mixed gently, and ice bath was carried out for 5 min; 150 ml of re-distilled phenol / chloroform (1:1) was added, the solution was mixed, and centrifugation was carried out at 12,000 rpm for 10 min; the upper aqueous phase was carefully aspirated into 2 volumes of pre-cooled absolute ethanol, and the solution was placed in a -20°C refrigerator for 2 hours; centrifugation was carried out at 12,000 rpm for 15 min, the ethanol was aspirated, 500 μl of 70% ethanol was carefully added to remove salts, and centrifugation was carried out to dryness; 18 μl of TE and 2 μl of RNAase enzyme solution were added, and incubation was carried out at 37°C for 1 hour; and the solution was used for enzyme digestion and further cloning.

[0155] 4.8) 20 ml, mix well and ice bath for 10 min; add equal volume of phenol: chloroform (1:1) solution and extract twice, add 2 volumes of pre-cooled absolute ethanol, and store at -20°C overnight, centrifuge at 12,000 rpm for 15 min, discard the supernatant, and wash the precipitate with 70% ethanol for 1-2 times, dry, dissolve in 3 ml of TE, add 10 μl of RNase solution (10 mg / ml), and incubate at 37°C for 30 min, and then pass through a Sepharose 2B column (1 X 10 cm, equilibrated with TE) to collect the first peak (large molecular weight DNA) using TE (pH 7.6) as eluent. Add 2 volumes of pre-cooled absolute ethanol and 1 / 10 volume of 3M NaAc (pH 5.2) to the collected sample, mix well, and store in a refrigerator at -20°C overnight, centrifuge at 12,000 rpm for 15 min, discard the supernatant, and wash the precipitate with 70% ethanol for 1-2 times, dry, dissolve in 1 ml of TE, and determine the DNA content by measuring the absorbance at 260 nm using a UV spectrophotometer. The large-scale extracted recombinant plasmid DNA can be used for the protoplast transformation of Pichia pastoris host cells according to the method of the present application and for long-term storage at low temperature.

[0156] (V) Preparation of E. coli competent cells and transformation of recombinant plasmid

[0157] Inoculate 2 ml of LB medium with E. coli host cells, and shake at 37°C overnight. The next day, inoculate 500 ml of LB medium with the overnight culture, and shake at 37°C at 300 rpm for 1 hour until the cell density reaches 0.5 OD600. Centrifuge at 5000 rpm for 5 min at 4°C, discard the supernatant, suspend the cells in 25 ml of cold solution containing 100 mM CaCl2and 10 mM Tris-HCl (pH 7.4), and store in an ice bath for 30-60 min. Then, centrifuge at 5000 rpm for 5 min at 4°C, carefully discard the supernatant, suspend the cells in 2 ml of calcium chloride cold solution, and store in an ice bath for 40 min. The cells can be used for transformation.

[0158] Suspend 200 μl of the above freshly prepared competent E. coli cells in an Eppendorf tube, add 10 μl of recombinant plasmid DNA ligation solution, and store in an ice bath for 30 min. Heat shock at 42°C for 2 min, add 500 μl of LB medium, and shake at 37°C for 1 hour. Centrifuge at 10,000 rpm for 10 sec, discard most of the LB, retain about 200 μl of LB, and mix the bacterial suspension gently with a gun tip. Divide the mixture into 50 μl and 150 μl, and coat each on a LB agar plate containing 50 μg / ml of ampicillin. Incubate in a 37°C incubator for 8-16 hours.

[0159] The preparation method of the present embodiment for efficiently obtaining high-purity recombinant human serum albumin comprises the following steps:

[0160] S1, preparing a new host strain, which is preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No. 30175 and a preservation date of March 28, 2024;

[0161] S2, fermentation;

[0162] S3, purification.

[0163] The new host strain is an engineering strain for expressing a recombinant HSA obtained by introducing a recombinant expression vector into a Pichia pastoris CBS7435 strain, and the nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO: 1.

[0164] The fermentation of step S2 specifically includes

[0165] S210, configuring a culture medium;

[0166] S220, electrode calibration;

[0167] S230, feeding;

[0168] S240, inoculation, inoculating the new host strain with high expression of human serum albumin;

[0169] S250, fermentation;

[0170] S260, tank release.

[0171] The recombinant expression vector comprises:

[0172] (1) a 5' regulatory region from Pichia pastoris, which contains a promoter element, and the 5' regulatory region is selected from the alcohol oxidase AOX1 gene, the dihydroxyacetone synthase DAS1 gene, or the histidinol dehydrogenase HIS4 gene from Pichia pastoris, and the 3' end of the regulatory region is connected to the sequence of (2) below;

[0173] (2) an optimized gene encoding human serum albumin, wherein the nucleotide sequence contained therein is shown in SEQ ID NO: 2, and the amino acid sequence contained therein is shown in SEQ ID NO: 3;

[0174] (3) a 3' termination sequence from Pichia methanolica, and the 3' termination sequence is selected from the 3' termination sequence of the AOX1 gene, the AOX2 gene, or the His4 gene from Pichia pastoris.

[0175] The nucleic acid sequence length of the optimized gene coding human serum albumin is 1755bp, and the corresponding coding amino acid sequence is 585 amino acids, which is one of the longest functional genes chemically synthesized in the world so far.

[0176] The present application relates to the specific and brand-new optimization design of HSA gene, which adopts three core databases of molecular biology: 1. International nucleic acid sequence database (GenBank / EMBL / DDBJ); 2. Swiss protein sequence and annotation database (Swiss-PROT); 3. Protein and biomolecule three-dimensional structure database provided by Brookhaven National Laboratory of the United States (Protein Data Bank, PDB). Various computer software packages (including GENESIS and PROSIS software packages prepared by Genetic Computer Group of the University of Wisconsin, Caltec software package prepared by California Institute of Technology, DNASIS and PROSIS software packages provided by Pharmacia Company of Sweden and other programs) are used for auxiliary analysis in multiple aspects, and finally the optimized gene coding human serum albumin is obtained.

[0177] The present application further relates to the optimized gene coding human serum albumin, which comprises the following components:

[0178] At least one marker gene available for screening in E. coli;

[0179] A DNA fragment of a replication origin capable of replicating in the E. coli host bacteria;

[0180] Further comprising at least two marker genes available for screening in yeast.

[0181] In the optimized gene coding human serum albumin, the preferred codon of the alcohol oxidase (AOX1) gene of Pichia pastoris is selected and controlled to account for 90% of the total number of codons of the coding optimized gene.

[0182] In the optimized gene coding human serum albumin, three restriction endonuclease sites of SalI, Hind III and Xba I are inserted in the optimized gene from 5' to 3' direction in sequence, so that the optimized gene is relatively evenly divided into four large fragments, facilitating the splicing, cloning and assembly of the synthesized gene.

[0183] In the optimized gene coding human serum albumin, the continuous G-C pairing is reduced, and the A-T pairing preferred by Pichia pastoris is increased, so that the GC content in the optimized gene is adjusted to 45-50%, and the optimal ratio of 47.64% is preferred in the embodiment, which is beneficial to the high expression of the target gene (see Fig. 1).

[0184] The optimized gene encoding human serum albumin further comprises an expression reading frame of

[0185] A 5' end restriction enzyme BamHI site is inserted into the 5' regulatory region (promoter region) of the AOX1 gene, followed by a CCAAACGATG 10 deoxy oligonucleotide (containing a Kozak sequence of a eukaryotic gene, i.e., AXXATG), followed by a Saccharomyces cerevisiae-derived yeast a-mating pheromone leader peptide (comprising 85 amino acids) sequence, and then a HSA mature gene is inserted between EcoRI and NotI of the multiple cloning site of the pPIC9K recombinant expression vector.

[0186] An encoding sequence AAAAGA of two double basic amino acids (-Lys-Arg-) is inserted after the EcoRI enzyme cutting site at the 5' end of the target gene, and the specific sequence is SEQ ID NO: 4; a double stop codon TAATAG is inserted before the NotI enzyme cutting site at the 3' end of the target gene, and the specific sequence is SEQ ID NO: 5. See FIG. 2; wherein an encoding sequence AAAAGA of two double basic amino acids (-Lys-Arg-) is inserted after the EcoRI enzyme cutting site at the 5' end of the target gene, for processing and cleavage by the endogenous KEX-2 protease in yeast; a double stop codon TAATAG is inserted before the NotI enzyme cutting site at the 3' end of the target gene, to strengthen the termination signal of translation and prevent the read-through phenomenon during gene expression.

[0187] The chemically wholly synthesized mature gene (1755 bp) encoding human serum albumin is cut from the pUC18 cloning vector by EcoRI-SmaI double digestion, and the fragment is identified by 1% agarose gel electrophoresis, and the results are shown in FIG. 3 (A: lane 1 represents the recombinant plasmid; lane 2 represents the recombinant plasmid digested with EcoRI and SmaI; M represents the molecular weight standard KB Ladder. B: two lanes respectively represent the molecular weight standard KB Ladder and DL3000), which is consistent with the expected results; the KpnI of the multiple cloning site of the pUC18 cloning vector is changed to NotI; the chemically wholly synthesized mature gene (1755 bp) encoding human serum albumin is cut from the pUC18 cloning vector by EcoRI-NotI double digestion, and the corresponding size gel strip is cut, and the fragment of the target gene is recovered by using a DNA recovery kit. The HSA fragment cut from the pUC18 cloning vector by double digestion of EcoRI-NotI is cloned into the corresponding double-digested Pichia pastoris recombinant expression vector pPIC9K to construct a recombinant expression vector HSA-pPIC9K (containing the HSA EcoRI-NotI fragment), and the specific recombinant vector flow chart is shown in FIG. 4, and the connection reaction system is as follows:

[0188] The ligation was performed overnight at 16°C in a 20 μl reaction system.

[0189] The ligation liquid containing the recombinant plasmid HSA-pPIC9K was added with 4 μl and 8 μl respectively to transform the competent E. coli JM109 host bacteria, and then spread on LB (containing ampicillin 20 μg / ml) plate and cultured in 37°C incubator overnight. Then, 18 colonies containing the recombinant plasmid HSA-pPIC9K were randomly picked up, inoculated in 2 ml LB (containing ampicillin 20 μg / ml) respectively, and shaken in a shaker for 6-8 hours. The plasmid double-stranded DNA was quickly extracted by alkali denaturation method, and then digested with EcoRI-NotI double enzyme respectively. The 1% agarose gel electrophoresis was performed to identify the recombinants containing HSA gene with corresponding size. Five clones containing HSA EcoRI-NotI fragment were identified. Then, the E. coli host bacteria containing the identified recombinant plasmid HSA-pPIC9K were inoculated, and a large amount of recombinant plasmid containing HSA-pPIC9K was extracted by large-scale plasmid extraction method for long-term preservation or subsequent transformation of Pichia pastoris host bacteria. The specific design details and sequencing results of the recombinant plasmid HSA-pPIC9K are shown in Figures 4 and 5, 6, 7.

[0190] The recombinant plasmid was linearized by using restriction enzymes SacI or Bgl II, and the linearized recombinant plasmid was used to transform Pichia pastoris host bacteria by electroporation method, i.e. to transform host bacteria GS115 or CBS7435. The specific implementation method is as follows:

[0191] a. Preparation of culture medium

[0192] 1. BMGY culture medium: yeast powder 10 g, peptone 20 g, dissolved in 700 mL deionized water, wet heat sterilized for 20 min, after cooling, add 100 mL 1M, pH 6.0, potassium phosphate buffer, 100 mL 10×YNB, 2 mL 500×B, 100 mL 10×GY, store at 4°C.

[0193] 2. BMMY culture medium: yeast powder 10 g, peptone 20 g, dissolved in 700 mL deionized water, wet heat sterilized for 30 min, after cooling, add 100 mL 1M pH 6.0 potassium phosphate buffer, 100 mL 10×YNB, 2 mL 500×B, 100 mL 10×M, store at 4°C.

[0194] 3. MD culture medium: add 100 mL 10×YNB, 2 mL 500×B, 100 mL 10×D to 800 mL sterilized water, store at 4°C.

[0195] 4. 1M, pH 6.0 potassium phosphate buffer: 132 mL 1M K2HPO4, 868 mL 1M KH2PO4, pH 6.0, autoclaved, stored at 4°C.

[0196] 5. 10 x YNB: YNB 134 g (with ammonium sulfate) dissolved in 1000 mL deionized water, filter sterilized, stored at 4°C.

[0197] 6. 500 x B: 20 mg Biotin dissolved in 100 mL deionized water, filter sterilized, stored at 4°C.

[0198] 7. 10 x M: 5 mL methanol mixed with 95 mL deionized water, filter sterilized, stored at 4°C.

[0199] 8. 10 x GY: 100 mL glycerol mixed with 900 mL deionized water, autoclaved, stored at 4°C.

[0200] 9. 10 x D: 100 g glucose dissolved in 1000 mL deionized water, filter sterilized, stored at 4°C.

[0201] 10. 1 M Sorbitol: 18.2 g D-sorbitol dissolved in 100 mL deionized water, filter sterilized, stored at 4°C

[0202] b. Cell preparation

[0203] 1. Pick a single colony of Pichia pastoris (GS115 or CBS7435) and inoculate into a 50 mL conical flask containing 5 mL YPD medium, incubate at 30°C, 250-300 r / min overnight;

[0204] 2. Take 100-500 μL of the culture and inoculate into a 2 L conical flask containing 500 mL fresh medium, incubate at 28-30°C, 250-300 r / min overnight, until OD600 reaches 1.3-1.5;

[0205] 3. Centrifuge the cell culture at 4°C, 1500 g for 5 min, resuspend the cell pellet with 500 mL ice-precooled sterile water;

[0206] 4. Centrifuge as in step 3, resuspend the cell pellet with 250 mL ice-precooled sterile water;

[0207] 5. Centrifuge as in step 3, resuspend the cell pellet with 20 mL ice-precooled 1 M sorbitol solution;

[0208] 6. Centrifuge as in step 3, resuspend the cell pellet with 1 mL ice-precooled 1 M sorbitol solution, the final volume is about 1.5 mL;

[0209] Note: The 80 μl aliquots can be frozen, but this will affect the transformation efficiency (within 2 weeks).

[0210] c. Electroporation

[0211] 1. Add 80 μL of the prepared Pichia pastoris competent cells and 5-20 μg of the linearized DNA (dissolved in double distilled water, 5-10 μL) into a 1.5 mL pre-chilled centrifuge tube, mix well. Then transfer the mixture into a pre-chilled transformation cuvette (0.2 cm type);

[0212] 2. Chill the transformation cuvette containing the transformation mixture for 5 min;

[0213] 3. According to the information provided by the electroporator, refer to other literatures and try many times, determine the appropriate voltage, current, capacitance and other parameters, according to the optimized parameters, carry out the electric shock transformation; the preferred parameters of the present application are: voltage 1500-1800 V; capacitance 25 μF; resistance 200-400 Ω, shock time 4-10 msec.

[0214] 4. Immediately after the pulse, add 1 mL of ice-bath 1 M sorbitol solution to the transformation cuvette, then transfer the transformation solution into a new 1.5 mL centrifuge tube;

[0215] 5. Incubate at 28 °C for 1-2 h on a shaker;

[0216] 6. Take 50-200 μL of the transformation solution of Pichia pastoris host strain GS115 or CBS7435 and spread on MD plates;

[0217] 7. Incubate in a 28 °C incubator for about 3-4 days until many single colonies containing recombinant plasmid positive colonies (yeast transformants) appear.

[0218] d. Screening of multi-copy transformants

[0219] 1. Use 2 mL of sterile water to rinse the above MD plate on which the yeast transformants grow, and then use a pipette to take the bacterial solution and temporarily store it in a centrifuge tube;

[0220] 2. Spread the obtained bacterial solution on YPD plates containing G418 resistance, 100 μL of bacterial solution per plate; wherein the G418 antibiotic content is configured into different concentration gradients: 0.25%, 0.5%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, etc.

[0221] 3. Place the plates containing the bacterial solution in a 28 °C incubator for 4-12 days to grow different G418-resistant white single colonies (i.e. obtain Pichia pastoris transformants with different copy numbers).

[0222] The experimental results of the host bacteria GS115 or CBS7435 by the electric transformation are shown in part in Figure 8, Figure 9, Figure 10, Table 1 and Table 2.

[0223] Table 1 Experimental results of the host bacteria GS115 by the electric transformation Note: In Table 1, the G418 resistance of 0.25 means that the host bacteria contains one copy of the exogenous gene, and so on.

[0224] Table 2 Experimental results of the host bacteria CBS7435 by the electric transformation Note: In Table 2, the G418 resistance of 0.25 means that the host bacteria contains one copy of the exogenous gene, and so on.

[0225] The transformants containing the recombinant plasmid HSA-pPIC9K with different copy numbers screened by the electric transformation are randomly picked from the YPD culture plate, and 6 clones are inoculated in 4 ml of BMGY liquid medium in a test tube. The test tube is shaken at 28°C and 200-250 rpm in a shaking bed overnight. The bacteria in the test tube are centrifuged at 1500-3000 g for 5 min at room temperature, and the bacteria are resuspended in 4 ml of BMMY with the original culture volume. The test tube is shaken at 28°C and 250-300 rpm in a shaking bed for 36-48 hours. The test tube is centrifuged at 5000 rpm for 10 min, and 30 μL of the supernatant is taken and dried by vacuum suction. The sample is subjected to 10% SDS-PAGE electrophoresis identification, and the expression product of the pPIC9K empty vector is used as a control to identify the high expression clones. Then, the high expression clones screened are preserved at low temperature by 15% glycerol, and used as the engineering strains for further expression experiments.

[0226] The pilot induction experiment is carried out for the above engineering high expression strain. If the strain is Pichia pastoris GS115, the experimental scheme is as follows: the glycerol bacteria are inoculated in YPD liquid medium for rejuvenation, 1 ml of the seed bacteria liquid is inoculated in 50 ml of BMGY culture solution, and the test tube is shaken at 28°C and 200-250 rpm for 18-24 hours. Then, 40 ml of the seed bacteria is inoculated in 1000 ml of BMGY culture medium at a 4% inoculation amount, and the test tube is shaken at 28°C and 250-300 rpm in a shaking bed for 24-30 hours. The culture solution is centrifuged at 5000 rpm for 5 min, and the supernatant is discarded. Then, the bacteria are suspended in 500 ml of BMMY induction expression medium, and the test tube is placed at 28°C and 250-300 rpm in a shaking bed for continuous shaking for 3-4 days. The 100% methanol is added every 24 hours to a final concentration of 0.5%. At this time, the cell density of the Pichia pastoris engineering strain is generally 18-20 OD 600Most of the expressed foreign proteins are secreted into the liquid culture medium. The fermentation medium was centrifuged at 4°C, 10000 rpm for 20 min, and the supernatant containing a large amount of HSA expression products was discarded. The supernatant sample was identified by SDS-PAGE electrophoresis. This small-scale induction experiment was used to analyze and identify the expression level of recombinant HSA.

[0227] Small-scale induction experiments were performed on the above-mentioned engineered high-expression strains. If it was Pichia pastoris CBS7435 strain, the experimental scheme would be as follows: glycerol strain→YPD→YPD→BSM→BSM: sample addition amount similar to GS115 strain.

[0228] This example is a small-scale induction experiment on part of Pichia pastoris GS115 or CBS7435 strain. Some results are shown in Figures 11, 12 and Figures 13, 14.

[0229] The engineered strains with obvious expression bands in the above-mentioned small-scale induction experiment were further subjected to high-cell-density culture fermentation test in 5L, 30L, 50L, 500L fermenters using fed-batch fermentation. The purpose was to explore the Pichia pastoris BSM fermentation process and further verify the pilot fermentation process of GS115 or CBS7435 engineered strains after electrotransformation. The specific implementation is as follows:

[0230] S210, configure the culture medium;

[0231] The fermentation medium of this example was prepared as shown in Table 3 below.

[0232] Table 3 Preparation of fermentation medium (prepared according to 6L volume)

[0233] When preparing the fermentation medium, the amount of phosphoric acid and glycerol was stored separately in a measuring cup or beaker. The other inorganic salts were dissolved in about 2L of purified water in sequence, i.e. after complete dissolution of the previous reagent, the next reagent was added. The last KOH also needed to be dissolved separately and not mixed with the inorganic salts.

[0234] S220, electrode calibration, including pH electrode calibration and dissolved oxygen electrode calibration.

[0235] pH electrode calibration: first prepare the standard pH electrode solution of pH 6.86 and 4.00, washing bottle and paper towel. Connect the pH electrode to the electrode line of the fermenter, rinse the electrode with the washing bottle, then absorb the water with the paper towel, insert the electrode into the pH 6.86 electrode solution, and when the pH value displayed by the fermenter is stable, press the calibration key to calibrate the 0 point. Take out the electrode, rinse it with the washing bottle, then absorb the water with the paper towel, insert it into the pH 4.00 electrode solution, and similarly when the displayed pH value is stable, press the calibration key to calibrate the slope. Repeat the operation 3 times, the pH display value should be consistent with the value of the electrode solution, that is, the calibration of the pH electrode is completed.

[0236] Dissolved oxygen electrode calibration: connect the dissolved oxygen electrode to the electrode line of the fermenter, and after cleaning, insert it into saturated anhydrous sodium sulfite solution, and after standing for 15 minutes, the dissolved oxygen display value should no longer change, at this time press the calibration key to calibrate the dissolved oxygen 0 point. The slope of the dissolved oxygen electrode is calibrated at 100% under the conditions of 100 rpm stirring and 0.5 vvm aeration, tank pressure 0.05 MPa before fermentation inoculation, and the calibration of the dissolved oxygen electrode is completed.

[0237] S230, feeding.

[0238] Feeding process: sequentially add phosphoric acid and glycerol into the fermenter, then add inorganic salt solution, finally add KOH, and add purified water to 5L (the reference tank weight is about 5Kg, after sterilization it should be about 6L). After the feeding is completed, the pH value should be below 2. Prepare PTM1 for a total of 1L. Store in a sealed and light-proof manner.

[0239] 100% analytical pure methanol 3L;

[0240] 50% glycerol feed 800ml:

[0241] Weigh 400g 98% glycerol, add purified water to 800ml, dissolve and pour into a 1L feed bottle, sterilize at 121℃ for 30 minutes, and cool for standby.

[0242] S240, inoculation, inoculate the new host strain with high expression of human serum albumin;

[0243] Pichia pastoris engineering strain fermentation experiment process

[0244] 1. Seed preparation:

[0245] Take the glycerol bacteria numbered CBS7435-C1-C50 or GS115-G1-G30, 100 μl to 20 ml of bacteria liquid to 100 ml of YPD medium as a primary seed liquid, 28°C shaking bed 200 rpm for 24 hours; transfer 5 ml of bacteria liquid to YPD 480 ml / 2L conical flask as a primary seed liquid, continue to cultivate at 28°C shaking bed 200 rpm for 24 hours. Transfer 5 ml of primary seed bacteria liquid to BSM medium 480 ml / 2L conical flask (total 2 bottles) as a secondary seed liquid, continue to cultivate at 28°C shaking bed 200 rpm for 24 hours. The OD 600 Should be between 20 and 40.

[0246] Inoculate the seed bacteria into the fermenter:

[0247] Before inoculation, confirm that the fermenter temperature is 30°C, set pH 5.0, automatically adjust pH with ammonia, and inoculate the seed liquid into the fermenter by flame circle inoculation. Flame circle inoculation, surround the inoculation port of the fermenter with an alcohol cotton ball and ignite. Close the small air inlet valve and open the large air outlet valve to about 0.02 MPa. Use a special tool to slightly open the inoculation port, and a small amount of exhaust to no air flow sound, then unscrew the inoculation port. Open the packaging rope of the seed bacteria bottle and the PTM1 container, remove the sealing film above the flame circle, and then pour the seed bacteria and PTM1 into the tube. Do not mix the seed bacteria and PTM1 before inoculation.

[0248] Recombinant human serum albumin fermentation culture stage:

[0249] (1) Engineering bacteria basic culture stage

[0250] Start the SCADA process control software for data recording, fermentation time recording and batch number recording, and open the atlas recording when the engineering bacteria fermentation starts. Record the relevant fermentation data (such as time, temperature, stirring, pH, DO, air flow, tank pressure) every 4 hours since the seed bacteria are inoculated into the fermenter. The control parameters are set as follows: control the pH at 5.5±0.1 with ammonia, and the temperature at 28±1°C; control the DO value above 30, the air flow at 2vvm, and the tank pressure at 0.03-0.04Mpa; after DO rebound (DO rebound refers to the increase of DO value by more than 10 under the condition that the fermenter is not ventilated, stirred, and tank pressure is not operated), enter the transition culture phase; when rebounding, take samples to detect OD 600 Should be around 50.

[0251] (2) Engineering bacteria transition culture stage:

[0252] The transition phase of the engineering bacteria is to further improve the biomass of the engineering bacteria. The fermentation data is recorded every hour. The transition phase stage needs to flow 50% glycerol: after the DO rebound, immediately flow 50% glycerol (add 12 ml / L of PTM1 solution containing biotin, about 10 ml); at the same time, adjust the stirring to the specified maximum speed of the fermenter, adjust the air flow to 2 vvm, and adjust the tank pressure to 0.04-0.08 Mpa; under this condition, the feeding speed is controlled to make the DO value at 30±10. At least once every 1 hour, DO-Spike is performed to ensure that the DO-Spike response time is below 60 seconds. The flow cycle is about 4-5 hours; when glycerol is added, the average pH set value is adjusted every 1 hour to make the pH after the end of glycerol feeding meet the project requirement of the induction pH value (in this fermentation, the induction stage pH and the culture stage pH are the same, both are 5.7, so there is no need to adjust the pH); when the transition phase of the engineering bacteria ends, the OD 600 is about 100. At this time, the wet weight of the bacterial solution is detected and the sample is taken 0h, and the wet weight is detected every time the sample is taken without detecting OD. Stop adding glycerol, and prepare to add methanol after the dissolved oxygen DO rebounds; add 4 g / L of methanol at one time (in this experiment, 24 g of methanol is added at one time, and 30 ml of methanol can be added through the feeding grommet using a disposable syringe). After the added methanol is consumed (DO rebounds), start adding methanol in the form of DO-Spike, and the DO-Spike response time is preferably below 60 seconds, and the DO value is controlled at 10-30; start adding methanol while adjusting the temperature to the project specified temperature (the induction temperature in this experiment is 24°C); control the pH at the project specified pH 5.7; every 12 hours, do DO-Spike and record the response time and other fermentation data; every 12 hours, take a sample to detect the pH and the wet weight of the engineering bacteria, centrifuge, take 100 μl of supernatant, add electrophoresis buffer, boil at 100°C for 5 minutes, and then store in -20°C; the other remaining supernatant is stored in a refrigerator at 2-8°C for detection or electrophoresis analysis; label the sample when taking the sample. During the induction expression phase of the Pichia pastoris engineering bacteria, the methanol concentration in the culture medium in the tank is maintained at 0.2-0.9%.

[0253] (3) Engineering bacteria induction expression phase:

[0254] After stopping the addition of glycerol, the dissolved oxygen DO rebounds, and preparation for adding methanol (add 12 ml / L of PTM1 in methanol); add 4 g / L of methanol at one time (in this experiment, 24 g of methanol is added at one time, and 30 ml of methanol can be added through the feeding grommet using a disposable syringe). After the added methanol is consumed (DO rebounds), start adding methanol in the form of DO-Spike, and the DO-Spike response time is preferably below 60 seconds, and the DO value is controlled at 10-30; start adding methanol while adjusting the temperature to the project specified temperature (the induction temperature in this experiment is 24°C); control the pH at the project specified pH 5.7; every 12 hours, do DO-Spike and record the response time and other fermentation data; every 12 hours, take a sample to detect the pH and the wet weight of the engineering bacteria, centrifuge, take 100 μl of supernatant, add electrophoresis buffer, boil at 100°C for 5 minutes, and then store in -20°C; the other remaining supernatant is stored in a refrigerator at 2-8°C for detection or electrophoresis analysis; label the sample when taking the sample. During the induction expression phase of the Pichia pastoris engineering bacteria, the methanol concentration in the culture medium in the tank is maintained at 0.2-0.9%.

[0255] (4) Tank release

[0256] The Pichia pastoris engineering strain tank fermentation experiment, the induction expression time of this experiment is about 200 hours, then tank release, solid-liquid separation and subsequent processing; and collect the supernatant for subsequent purification.

[0257] The detection of the target protein in the fermentation test process of the Pichia pastoris engineering bacteria is as follows:

[0258] In this embodiment, the fermentation process of the Pichia pastoris engineering bacteria in the fermenter is controlled on line and recorded in real time, and in particular, the target protein in the fermentation broth in the fermenter is tracked and detected on line at different time nodes in the induction expression stage. The BCA protein concentration determination kit (enhanced) of the Biyun Tian Biotechnology Company is used as a detection tool in the present application, and the BCA protein concentration determination kit (enhanced) is developed according to the BCA method which is one of the two most commonly used protein concentration detection methods in the world, and high stability, high sensitivity and high compatibility of the protein concentration determination results are achieved. In this experiment, bovine serum albumin is used as a standard, a standard curve and a linear equation are established (see FIG. 18), and the expression amount of the HSA secreted and expressed in each batch of fermentation broth in the fermenter is quantitatively analyzed; the specific method is that at different time nodes in the induction expression phase, the fermentation broth in the fermenter is sampled, and the fermentation sample is centrifuged (5000g x 20min), the bacterial body is discarded, and the supernatant is retained; after the fermentation of this batch is completed, the retained fermentation supernatant sample is subjected to SDS-PAGE electrophoresis, and then subjected to staining with coomassie brilliant blue, and the electrophoresis result is observed, as shown in FIG. 15, FIG. 16 and FIG. 17; on the other hand, the retained fermentation supernatant sample is diluted, a 96-well plate is used for sampling and a color developing agent is added, and the sample is detected according to the instruction of the BCA protein concentration determination kit (enhanced), and the expression level of human serum albumin at different time nodes in the fermentation process is calculated, and part of the results are shown in Table 4 and Table 5. In addition, the expression amount of the recombinant HSA in the results of 9 batches of fermentation experiments is shown in Table 6.

[0259] Table 4 Determination values of the target protein concentration in the supernatant at different time nodes in the fermentation test of the engineering strain C7 in a 50L fermenter

[0260] Table 5 Determination values of the target protein concentration in the supernatant at different time nodes in the fermentation test of the engineering strain C1 in a 50L fermenter

[0261] Table 6 Experimental results of producing gene recombinant human serum albumin by high-density fermentation technology

[0262] In Table 6, C represents the CBS7435 engineering bacteria, and G represents the GS115 engineering bacteria

[0263] The step S300 is specifically as follows:

[0264] S310, taking out the fermentation broth;

[0265] S320, centrifuging the fermentation broth;

[0266] S330, membrane filtering the supernatant;

[0267] S340, heat treating;

[0268] S350, secondary membrane filtering;

[0269] S360, chromatography;

[0270] The step S310 specifically comprises taking the fermentation broth from the fermenter after the fermentation is completed in step S2.

[0271] The step S320 specifically comprises centrifuging the fermentation broth taken in step S310 at 9000 rpm, with temperature control below 30℃.

[0272] The step S330 specifically comprises filtering the fermentation supernatant after centrifugation in step S320 by hollow fiber membrane to obtain the filtered fermentation supernatant.

[0273] The step S340 specifically comprises adding sodium octanoate to the filtered fermentation supernatant obtained in step S330, so that the final concentration of sodium octanoate is 5 mmol / L, adjusting the pH value to 5.8-7.0, and heating at 68℃ for 30 min; quickly cooling, and then adjusting the pH value to 4.5 with acetic acid to obtain the heat-treated fermentation supernatant.

[0274] The step S350 specifically comprises filtering the heat-treated fermentation supernatant obtained in step S340 by hollow fiber membrane again to obtain the fermentation supernatant to be purified.

[0275] The step S360 specifically comprises chromatographing the fermentation supernatant to be purified obtained in step S350 to obtain the purified genetically recombined human serum albumin.

[0276] The column chromatography is selected from

[0277] TH-MC complex mode SP cation exchange column chromatography and hydroxyapatite type II (HAP II) column chromatography and DEAE anion exchange column chromatography, etc.

[0278] or

[0279] TH-MC complex mode SP cation exchange column chromatography and hydroxyapatite type II (HAP II) column chromatography and TA-phenyl-HIC hydrophobic column chromatography, etc.

[0280] The purified recombined human serum albumin sample is subjected to gel electrophoresis, and the gel electrophoresis shows a single band, as shown in FIG. 19. The purity of the genetically recombined human serum albumin sample is 99.03% as shown by HPLC liquid phase detection, and the results are shown in FIG. 20.

[0281] The purified recombinant human serum albumin sample (No. P7901-PH-E) and the rHSA national standard sample (purchased from the China Food and Drug Administration Inspection Research Institute) were subjected to preliminary research on the mass spectrometry molecular weight characterization method, and comparative research was conducted under non-reducing conditions and reducing conditions using mass spectrometry-SEC, RP elution conditions. The results showed that the total ion chromatogram (TIC) chromatogram under the above conditions showed a single peak, and the peak time and peak shape were highly consistent (see Figures 21 and 22). Further experiments were conducted using electrospray mass spectrometry to determine the molecular weight. The results showed that the recombinant human serum albumin sample (No. P7901-PH-E) provided by our company and the rHSA national standard sample had a reducing molecular weight mass spectrometry determination result of 66477.0 and 66487.5 Da, respectively, which was highly consistent with the theoretical molecular weight of HSA, which was 66472 Da, as shown in Figure 23.

[0282] The present embodiment relates to a recombinant expression vector for preparing high-expression human serum albumin, a novel host strain and its application. The recombinant expression vector and the novel host strain constructed have the following beneficial effects:

[0283] 1) The target gene is optimized at the molecular level to obtain the most suitable expression of the target gene in Pichia pastoris; 2) At the cellular level, the latest Pichia pastoris expression system host strain CBS7435 is introduced for gene expression of human serum albumin; the G418 resistance gene is used to screen Pichia pastoris transformants containing high copy number of foreign genes, which are used to construct Pichia pastoris engineering strains for high-level expression of recombinant human serum albumin; 3) At the engineering level, the expression level of the target protein is further improved by optimizing the fermentation culture process and reactor process control scheme; 4) At the purification process, the serious defects existing in the existing purification process of recombinant human serum albumin are analyzed and learned from the latest purification media, purification technology and purification methods at home and abroad. The present application only uses fermentation broth solid-liquid separation, heat treatment and ultrafiltration concentration for pretreatment, and then completes the entire purification process through three-step column chromatography. The purity of the recombinant human serum albumin reaches more than 99%, and the recovery rate of the entire purification process reaches more than 60%.

[0284] The above embodiments only express certain embodiments of the present application, which are described in more detail and in more detail, but should not be construed as limiting the scope of the present patent; it should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application; therefore, the protection scope of the present patent should be subject to the appended claims.

[0285] The sequences to which the invention relates:

Claims

1. A recombinant expression vector for preparing high expression of human serum albumin, characterized in that: The nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO:

1.

2. The recombinant expression vector for preparing high expression of human serum albumin according to claim 1, wherein, The recombinant expression vector comprises: ​ (1) a 5' regulatory region from Pichia pastoris, which contains a promoter element, and the 5' regulatory region is selected from the alcohol oxidase AOX1 gene, dihydroxyacetone synthase DAS1 gene or histidinol dehydrogenase HIS4 gene from Pichia pastoris, and the 3' end of the regulatory region is connected to the sequence of (2) below; (2) an optimized gene encoding human serum albumin, wherein the nucleotide sequence contained therein is shown in SEQ ID NO: 2, and the amino acid sequence contained therein is shown in SEQ ID NO: 3; (3) a 3' termination sequence from Pichia methanolica, and the 3' termination sequence is selected from the 3' termination sequence of the AOX1 gene, AOX2 gene or His4 gene from Pichia pastoris.

3. The recombinant expression vector for preparing high expression of human serum albumin according to claim 2, characterized in that: Further comprising at least one marker gene for screening in E. coli; a DNA fragment of a replication origin that can replicate in an E. coli host; Further comprising at least two marker genes for screening in yeast.

4. The recombinant expression vector for preparing high expression of human serum albumin according to claim 2, wherein the vector is pPIC9K-HSA. The recombinant expression vector is any one of pPIC9, pPIC3, pPICZαABC, pPIC3.5K, pHIL-S1, pHIL-D2, pA0804, pA0815, pGAPZαABC, pPIC6αABC, pPIC9K linearized by restriction endonuclease SacI or BglII.

5. The recombinant expression vector for preparing high expression of human serum albumin according to claim 4, wherein the vector is pPIC9K-HSA. The recombinant expression vector is HSA-pPIC9K linearized by restriction endonuclease SacI or BglII, which contains at least one copy of the optimized gene encoding human serum albumin.

6. The recombinant expression vector for preparing high expression of human serum albumin according to claim 1, wherein the vector is pPIC9K-HSA. In the optimized gene encoding human serum albumin, the preferred codon of Pichia pastoris alcohol oxidase (AOX1) gene is selected and the proportion of the preferred codon in the total number of codons of the optimized gene is 90%.

7. The recombinant expression vector for preparing high expression of human serum albumin according to claim 2, wherein the vector is pPIC9K-HSA. In the optimized gene encoding human serum albumin, three restriction endonuclease sites of SalI, Hind III and Xba I are inserted in the optimized gene from 5' to 3' direction, so that the optimized gene is relatively evenly divided into four large fragments.

8. The recombinant expression vector for preparing high expression of human serum albumin according to claim 2, wherein the vector is pPIC9K-HSA. In the optimized gene encoding human serum albumin, the continuous G-C pairing is reduced and the A-T pairing preferred by Pichia pastoris is increased, so that the GC content in the optimized gene is adjusted to 45-50%.

9. The recombinant expression vector for preparing high expression of human serum albumin according to claim 2, wherein the vector is pPIC9K-HSA. In the optimized gene encoding human serum albumin, the expression reading frame is The 5' end restriction endonuclease BamHI site is inserted into the 5' regulatory region (promoter region) of the AOX1 gene, followed by CCAAACGATG 10 deoxyoligonucleotides (containing the Kozak sequence of eukaryotic genes, i.e. AXXATG), followed by the sequence of the Saccharomyces cerevisiae-derived yeast a-mating pheromone leader peptide (composed of 85 amino acids), and then the HSA mature gene is inserted between EcoRI and NotI of the multiple cloning site of the pPIC9K recombinant expression vector. The coding sequence AAAAGA of two double basic amino acids (-Lys-Arg-) is inserted after the EcoRI enzyme cutting site at the 5' end of the target gene, and the specific sequence is SEQ ID NO: 4; and a double termination codon TAATAG is inserted before the NotI enzyme cutting site at the 3' end of the target gene, and the specific sequence is SEQ ID NO:

5.

10. A novel host strain for high expression of human serum albumin, characterized in that: The new host strain is Pichia pastoris HSA-C16, which is preserved in the China General Microbiological Culture Collection Center on March 28, 2024, and the preservation number is CGMCC No. 30175.

11. The novel host organism expressing human serum albumin in high level according to claim 10, characterized in that: The new host strain is an engineering strain of an expression gene recombinant HSA obtained by introducing the recombinant expression vector for preparing high-expression human serum albumin according to any one of claims 1-9 into the Pichia pastoris CBS7435 strain.

12. Use of the novel host bacteria of high expression of human serum albumin according to claim 10, characterized in that: The new host strain is used in the preparation of human serum albumin.

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