Method for constructing new host bacterium with high expression of human serum albumin

Abstract

The present invention relates to a method for constructing a host bacterium with high expression of human serum albumin, which method comprises: S100, designing an optimized gene encoding human serum albumin; S200, constructing a recombinant expression vector; and S300, transforming the recombinant expression vector into a Pichia pastoris host bacterium. The optimization design of a full-length sequence of a target gene is performed at the molecular level to obtain a target gene with the most suitable expression in Pichia pastoris; at the cellular level, the latest international Pichia pastoris expression system host bacterium CBS7435 is introduced for the gene expression of human serum albumin; and Pichia pastoris transformants harboring high-copy-number exogenous genes are selected by means of using a G418 resistance gene for the construction of an engineering bacterium with high expression of recombinant human serum albumin.

Description

Construction method of novel host bacteria for high expression of human serum albumin 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 construction method of novel host bacteria for high expression of human serum albumin. BACKGROUND

[0002] The development of molecular biology technology provides many methods and means for producing exogenous proteins by using biological reactors. 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 three decades of development, such as host bacteria 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 target genes in the host genome, effective secretion and moderate glycosylation of expression products, 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, etc. 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, it is very suitable for pilot scale-up and expansion for industrial scale production.

[0003] The general steps for expressing foreign genes in Pichia pastoris include: 1) inserting foreign genes into Pichia pastoris recombinant expression vectors; 2) linearizing the recombinant plasmids by endonuclease treatment and 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 yeast genome; 6) identifying the expression level of foreign genes by small-scale induction expression; and 7) using bioreactors for large-scale fermentation production 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 the 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 in itself; further investigation shows that there are still many variables between high copy Pichia pastoris transformants and high expression of foreign proteins, and different optimal copy numbers are presented for different target genes, and there is no linear relationship between them. In the commonly used series of recombinant expression vectors pIC9K, pIC3.5K, etc. for Pichia pastoris, the G418 resistance gene is introduced. After transforming the host bacteria, the foreign gene and the resistance gene are integrated into the Pichia pastoris chromosome by homologous recombination. By increasing the G418 resistance screening, the transformants containing multiple copies of the target gene 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 gradients. The concentration gradients of G418 in the pre-experiment are 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 gradients, and to print the positive clones on the HIS4 auxotrophic plates one by one on the G418 gradient plates by photocopying to screen the high copy number clones. The characteristic of this method is that the workload is huge (the concentration of each single transformant is equivalent before photocopying, and three continuous subcultures are needed before photocopying to ensure the concentration), 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 gradients, but the difference is that all the positive transformants grown on the HIS4 auxotrophic plates 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 plates with different concentration gradients. 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, pre-experiments need to be carried out to explore the dilution degree 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 of G418 is large, the operation is complicated, the workload is large, and it is 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-scale 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, and the purity requirement is 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 construction method of the novel host strain for high expression of human serum albumin provided by the present application comprises

[0010] S100, designing an optimized gene encoding human serum albumin;

[0011] S200, constructing a recombinant expression vector;

[0012] S300, transforming the recombinant expression vector obtained in step S200 into a Pichia pastoris host strain by electroporation to obtain the novel host strain.

[0013] Preferably, the optimized gene encoding human serum albumin in step S100 comprises a nucleotide sequence as shown in SEQ ID NO: 2, and an amino acid sequence as shown in SEQ ID NO: 3.

[0014] Preferably, in the optimized gene encoding human serum albumin in step S100, the biased codons of the Pichia pastoris alcohol oxidase (AOX1) gene are selected and controlled to account for 90% of the total number of codons in the optimized gene.

[0015] Preferably, in the optimized gene encoding human serum albumin in step S100, 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.

[0016] Preferably, in the optimized gene encoding human serum albumin in step S100, 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%.

[0017] Preferably, in the optimized gene encoding human serum albumin in step S100, the expression reading frame also comprises

[0018] 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, a 10-deoxy oligonucleotide containing the Kozak sequence of eukaryotic genes (AXXATG), followed by the sequence of the Saccharomyces cerevisiae-derived a-mating pheromone leader peptide (consisting 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;

[0019] 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.

[0020] Preferably, the step S200 of constructing the recombinant expression vector specifically comprises

[0021] The chemically totally synthesized optimized gene encoding human serum albumin is cut off from the cloning vector by EcoRI-NotI double enzyme digestion, and the cut-off corresponding size gel strip is recovered by DNA recovery Kit to recover the fragment of the target gene; the fragment containing the target gene is cloned into the recombinant expression vector subjected to corresponding double enzyme digestion, so that the recombinant expression vector is constructed.

[0022] Preferably, the step S300 specifically comprises linearizing the recombinant expression vector obtained in the step S200 by using the restriction enzyme SacI or Bgl II, and linearizing the recombinant expression vector by using the method of electroporation to transform the Pichia pastoris host strain, so that the novel host strain is obtained.

[0023] Preferably, the nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO: 1.

[0024] Preferably, the recombinant expression vector is any one of pPIC9, pPIC3, pPICZ alpha ABC, pPIC3.5K, pHIL-S1, pHIL-D2, pA0804, pA0815, pGAPZ alpha ABC, pPIC6 alpha ABC, and pPIC9K.

[0025] Preferably, the recombinant expression vector is HSA-pPIC9K.

[0026] Preferably, the Pichia pastoris host strain is Pichia pastoris CBS7435.

[0027] Preferably, the novel host strain 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.

[0028] 3. Beneficial effects

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

[0030] The application discloses a construction method of a novel host bacterium for high expression of human serum albumin, and the construction method comprises the following steps: S100, designing an optimized gene for coding human serum albumin; S200, constructing a recombinant expression vector; and S300, transforming the recombinant expression vector obtained in the step S200 into a Pichia pastoris host bacterium by an electrotransformation method to obtain the novel host bacterium. The gene is optimized at a molecular level to obtain a gene suitable for expression of the Pichia pastoris, and the latest Pichia pastoris expression system host bacterium CBS7435 is introduced at a cell level to be used for gene expression of human serum albumin. The G418 resistance gene is used to screen the Pichia pastoris transformant containing a high copy number of exogenous genes, and the novel engineering bacterium for high-level expression of recombinant human serum albumin can be used for preparing human serum albumin with a purity of more than 99%.

[0031] The Pichia pastoris HSA-C16 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 28, 2024, and the address of the CGMCC is No. 1, Yabian West Road, Beijing City, Chaoyang District, No. 3, 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 the adjustment of the G-C pairing content in the nucleotide sequence of the optimized gene of the embodiment to the optimal ratio;

[0033] FIG. 2 is a pPIC9K map of the Pichia pastoris multi-copy recombinant expression vector 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 construction process schematic diagram 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 change of the cell state of the Pichia pastoris host bacterium by the electrotransformation of the recombinant plasmid;

[0040] FIG. 9 is a schematic diagram of the change of the cell state of the Pichia pastoris GS115 host bacterium by the electrotransformation of the recombinant plasmid;

[0041] Figure 10 is a schematic diagram of the cell state change of the recombinant plasmid to the Pichia pastoris CBS7435 host cell by electroporation in the embodiment;

[0042] Figure 11 is a first results diagram of the small-scale induction experiment of the Pichia pastoris GS115 engineering strain prepared in the embodiment;

[0043] Figure 12 is a second results diagram of the small-scale induction experiment of the Pichia pastoris GS115 engineering strain prepared in the embodiment;

[0044] Figure 13 is a first results diagram of the small-scale induction experiment of the Pichia pastoris CBS7435 engineering strain prepared in the embodiment;

[0045] Figure 14 is a second results diagram of the small-scale induction experiment of the Pichia pastoris CBS7435 engineering strain prepared in the embodiment;

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

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

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

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

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

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

[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 embodiment 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 embodiment 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 embodiment 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 of ordinary skill 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 shown in the drawings. These terms are mainly used to better describe the present application and its embodiments, and are not intended 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 partial terms can also be used to indicate other meanings, for example, the term "upper" can also be used to indicate a certain attachment relationship or connection relationship in some cases. For those of ordinary skill 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 a monolithic 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 of ordinary skill 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 present embodiment is a construction method of a new host strain for high expression of human serum albumin, and the construction method comprises

[0063] S100, designing an optimized gene coding human serum albumin;

[0064] S200, constructing a recombinant expression vector;

[0065] S300, transforming the recombinant expression vector obtained in step S200 into a Pichia pastoris host strain by an electrotransformation method to obtain the new host strain.

[0066] The nucleotide sequence contained in the optimized gene coding human serum albumin in step S100 is shown in SEQ ID NO: 2, and the amino acid sequence contained therein is shown in SEQ ID NO: 3.

[0067] In the optimized gene coding human serum albumin in step S100, the biased codons of the alcohol oxidase (AOX1) gene of Pichia pastoris are selected and controlled to account for 90% of the total number of codons of the optimized gene.

[0068] In the optimized gene coding human serum albumin in step S100, 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.

[0069] In the optimized gene coding human serum albumin in step S100, 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%.

[0070] Preferably, the optimized gene coding human serum albumin in step S100 further comprises an expression reading frame of

[0071] The restriction endonuclease 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 a-mating pheromone leader peptide (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.

[0072] 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; the 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.

[0073] The step S200 of constructing the recombinant expression vector is specifically constructing the recombinant expression vector by

[0074] The chemically fully synthesized optimized gene encoding human serum albumin is cut off from the cloning vector by EcoRI-NotI double digestion, and the cut corresponding size gel strip is recovered by DNA recovery Kit to recover the fragment of the target gene; the fragment containing the target gene is cloned into the recombinant expression vector which is cut by the corresponding double enzyme, and the recombinant expression vector is constructed.

[0075] The step S300 is specifically linearizing the recombinant expression vector obtained in step S200 by using restriction enzyme SacI or Bgl II, and the linearized recombinant expression vector is transformed into the Pichia pastoris host strain by using the method of electroporation, so as to obtain the novel host strain.

[0076] The nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO: 1.

[0077] The recombinant expression vector is any one of pPIC9, pPIC3, pPICZαABC, pPIC3.5K, pHIL-S1, pHIL-D2, pA0804, pA0815, pGAPZαABC, pPIC6αABC, pPIC9K.

[0078] The recombinant expression vector is HSA-pPIC9K.

[0079] The Pichia pastoris host strain is Pichia pastoris CBS7435

[0080] The construction process and experimental data of the novel host strain for high expression of human serum albumin in the embodiment are as follows:

[0081] The materials and equipment used include:

[0082] I. Strains

[0083] 1. Pichia pastoris host strain GS115 (Pichia pastoris (his4) NRRL Y-15851);

[0084] 2. Pichia pastoris host strain CBS7435 (Pichia pastoris NRRL Y-11430);

[0085] 3. E. coli host strain:

[0086] E. coli JM109 F' [(recAl, supE44 endAl hsdR17 gyrA96 relAl thi△(lac-proAB)

[0087] F' [traD36 proAB* lacIa lacZ△M15]);

[0088] E. coli HB101 (supE44 hsd S20(rB-mB-) recA: ara-14 proA2 lac Y: galK2 rpsL20 xyl-5 mtl-1).

[0089] The above E. coli host strains are used for cloning of target genes and construction and preparation of plasmids.

[0090] II. Main reagents

[0091] 1. DNA restriction enzymes, T4 DNA ligase, polymerase, etc. are purchased from GIBCO-BRL, Pharmacia, Bio-Labs and Huamei Bioengineering Co. Ltd. respectively;

[0092] 2. Casein hydrolysate (product of MERK, Germany);

[0093] 3. Bacto-yeast extract (product of Difco, USA);

[0094] 4. PCR amplification kit (product of Pharmacia, Sweden);

[0095] 5. DNA sequence analysis kit (product of USB, USA);

[0096] 6. Acrylamide (Acr), N, N-dimethyl bisacrylamide (Bis), sodium dodecyl sulfate (SDS), guanidine hydrochloride, urea, TEMED (product of Sigma, UK);

[0097] 7. IPTG, X-gal, DTT, Agarose (product of Sigma, UK);

[0098] 8. YNB, Biotin, Agar (product of Difco, USA);

[0099] 9. Sorbitol, glucose, L-histidine, L-lysine, L-methionine, L-leucine, L-isoleucine, L-glutamic acid (Sigma, USA);

[0100] 10. Glycerol, methanol (Shanghai Chemical Reagent Factory);

[0101] 11. Enzyme reaction solution:

[0102] Restriction enzyme high-salt buffer: 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2;

[0103] Restriction enzyme medium-salt buffer: 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl2;

[0104] Restriction enzyme low-salt buffer: 10 mM Tris-HCl (pH 8.0), 10 mM MgCl2;

[0105] T4 DNA ligase buffer: 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 10 mM DTT, 1 mM ATP;

[0106] 13. SDS-PAGE protein electrophoresis reagent:

[0107] Electrophoresis buffer: 192 mM glycine, 25 mM Tri-HCl, 0.1% SDS, pH 8.3;

[0108] Concentrated gel buffer: 125 mM Tris-HCl, 0.1% SDS, pH 6.8;

[0109] Separation gel buffer: 375 mM Tris-HCl, 0.1% SDS, pH 8.8;

[0110] Sample buffer (1x): 50 mM Tris-HCl (pH 6.8), 1% SDS, 10% glycerol, 2.5% mercaptoethanol, 0.05% bromophenol blue;

[0111] 30% Acrylamide: 29% acrylamide, 1% N, N-dimethyl bisacrylamide;

[0112] Coomassie brilliant blue staining solution: 0.25% (W / V) Coomassie brilliant blue G-250, 5% Hac;

[0113] 45% Ethanol decolorizing solution: 7.5% Hac, 10% ethanol;

[0114] 13. Commonly used buffers:

[0115] TE buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA;

[0116] STE buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 20 mM NaCl PBS buffer: 10 mM NaH2P04-NaHPO3(pH 7.0), 150 mM NaCl;

[0117] 10X TBS buffer: 108 g Tris base, 55 g boric acid, 40 ml EDTA (0.5 M) per liter, pH 8.0;

[0118] 50X TAE buffer: 242 g Tris base, 57.1 ml glacial acetic acid, 100 ml EDTA (0.5 M) per liter, pH 8.0;

[0119] Saturated phenol: saturated with TE buffer after redistillation, 20 ml aliquots stored at 20°C;

[0120] Phenol: chloroform: isoamyl alcohol (V / V): 1:1:0.8;

[0121] Chloroform: isoamyl alcohol (V / V): 24:1.

[0122] III. Main instruments and equipment

[0123] 1. ABI 381A DNA automatic synthesizer, product of American Applied Biosystems (ABI) Company;

[0124] 2. 5 liter RIBE-5 full-automatic fermentation tank, product of Shanghai Guoqiang Biochemical Engineering Equipment Co., Ltd.;

[0125] 3. 15 liter Biocenter 15F full-automatic fermentation tank, product of Shanghai Guoqiang Biochemical Engineering Equipment Co., Ltd. (software system: Fermentation Star);

[0126] 4. 50 liter Biocenter 50F full-automatic fermentation tank, product of Shanghai Guoqiang Biochemical Engineering Equipment Co., Ltd. (software system: Fermentation Star);

[0127] 5. 100 liter Biocenter 100F full-automatic fermentation tank, product of Shanghai Guoqiang Biochemical Engineering Equipment Co., Ltd. (software system: Fermentation Star);

[0128] 6. 500 liter full-automatic Bplus bioreactor, product of Germany B. Braun Company;

[0129] 7. SDL-100 protein purification system, Suzhou Saimu Instrument Co., Ltd. (medium pressure chromatography workstation, software SCG);

[0130] 8. Agilent high-performance liquid chromatograph, Agilent Technologies, 1260 infinity, product of Agilent Technologies, USA;

[0131] 9. External pressure ultrafiltration membrane module (filtration precision: 30KD), product of Huzhou Kelu Membrane Technology Co., Ltd.;

[0132] 10. External pressure ultrafiltration membrane module (filtration precision: 100KD), product of Huzhou Kelu Membrane Technology Co., Ltd.;

[0133] 11. Full-wavelength enzyme marker, thermo Fisher 1510, product of Thermo Fisher Scientific, USA;

[0134] 12. Electric shock conversion instrument, Micropulser411BR10654, USA BIO-RAD company.

[0135] Four, experimental method

[0136] (I) Culture medium and culture conditions

[0137] E. coli culture medium LB (1% bacto-typtone, 0.5% bacto-yeast extract, 1% NaCl), solid culture medium plus Agar powder 15 g / L, E. coli was cultured at 37°C; Pichia rich culture medium YPD (1% yeast extract, 2% peptone, 2% dextrose); Pichia protoplast regeneration culture medium RBD [1M sorbital, 1% dextrose, 1.34% YNB, 4×10 5 Biotin, 0.005% amino acid mixture (including L-glutamic acid, L-methionine, L-leucine, L-isoleucine, L-lysine); if it is solid culture medium, add 2% Agar powder in liquid culture medium; Pichia shake flask culture medium BMGY and BMMY (1% yeast extract, 2% peptone, 100mM PBS buffer, pH 6.0, 1.34% YNB, 4X105% Biotin, 1% glycerol or 0.5% methanol). Pichia was cultured at 30°C.

[0138] (II) One of the high-density fermentation culture medium and culture conditions of Pichia fermenter:

[0139] A, 10×Basal Salts:

[0140] 1. H3PO4, 85% 42 ml

[0141] 2. CaSO2-2H2O 1.8 g / L

[0142] 3. K2SO4 28.6 g / L

[0143] 4. MgSO4-7H2O 23.4 g / L

[0144] 5. KOH 6.5 g / L

[0145] B. 250 x PTMl salts:

[0146] 1. CuSO4-5H2O 6 g / L

[0147] 2. KI 0.08 g / L

[0148] 3. MnSO4-H2O 3 g / L

[0149] 4. Na2MoO4-2H2O 0.2 g / L

[0150] 5. H3BO3 0.02 g / L

[0151] 6. CoCl2 0.5 g / L

[0152] 7. ZnCl2 20 g / L

[0153] 8. FeSO4-7H2O 65 g / L

[0154] 9. Biotin 0.2 g / L

[0155] 10. H2SO4 5 ml

[0156] C. glycerol 8% (V / V)

[0157] D. Feed medium 50% glycerol (1 L) + 12 ml / L PTM1

[0158] E. Induced medium 100% methanol (1 L) + 12 ml / L PTM1

[0159] F. Dissolved O2(DO) >20%

[0160] G. pH 5.0-5.8

[0161] H. Temperature 20-30°C

[0162] (Three) Pichia pastoris fermenter high-density fermentation medium and culture conditions two:

[0163] A liquid preparation:

[0164] YPD medium (50 ml):

[0165] 1. Yeast extract 0.5 g

[0166] 2. Peptone 1 g

[0167] 3. Glucose 1 g

[0168] The above YPD medium is used for the culture of Pichia pastoris primary seed bacteria.

[0169] B liquid preparation:

[0170] BSM medium (1 L):

[0171] 1. 98% glycerol 63 g / L

[0172] 2. ddH2O (ultra-pure water) 912 ml

[0173] 3. CaSO2·2H2O 0.46 g / L

[0174] 4. MgSO4·7H2O 5.84 g / L

[0175] 5. K2SO4 7.34 g / L

[0176] 6. (NH4)2SO4 9 g / L

[0177] The above BSM medium is used for cultivation of Pichia pastoris secondary seed culture and as the main nutrient medium for yeast cultivation in bioreactor.

[0178] Preparation of C solution:

[0179] Preparation of sodium hexametaphosphate (HMP) solution (150 ml):

[0180] 1. (NaPO3)6 (Sodium hexametaphosphate) 30 g

[0181] 2. ddH2O (ultrapure water) 150 ml

[0182] Preparation of D solution:

[0183] PTM1 solution (1 L):

[0184] 1. CuSO4-5H2O 6 g / L

[0185] 2. KI 0.08 g / L

[0186] 3. MnSO4-H2O 3 g / L

[0187] 4. Na2MoO4-2H2O 0.2 g / L

[0188] 5. H3BO3 0.02 g / L

[0189] 6. CoCl2 0.5 g / L

[0190] 7. ZnCl2 20 g / L

[0191] 8. FeSO4-7H2O 65 g / L

[0192] 9. Biotin 0.2 g / L

[0193] 10. H2SO4 5ml

[0194] (iv) Extraction of plasmid

[0195] 1. Small scale plasmid extraction

[0196] The single colony was inoculated into 2ml LB medium containing the corresponding antibiotic and incubated at 37°C overnight. Next day, 1.5ml bacterial solution was taken into an Eppendorf tube and centrifuged to precipitate the bacterial cells. The supernatant was removed. The bacterial precipitate was placed in an ice bath and 100μl solution I (50mM Glucose, 25mM Tris-HCl, 10mM EDTA, pH 8.0) was added. The mixture was shaken and incubated at room temperature for 5min. Then 200μl solution II (0.2N NaOH, 1% SDS) was added and the mixture was shaken and incubated in an ice bath for 5min. Then 150μl solution III (3M NaAc, pH 4.8) was added and the mixture was shaken and incubated in an ice bath for 5min. Then 450μl phenol / chloroform (1:1) was added and the mixture was shaken and centrifuged at 12,000rpm for 10min. The upper aqueous phase was carefully taken and added to 2 times volume of pre-cooled absolute ethanol in an ice bath at -20°C for 2 hours. The mixture was centrifuged at 12,000rpm for 15min. The ethanol was removed and 500μl 70% ethanol was added to wash away the salt. After centrifugation, 18μl TE and 2μl RNAase enzyme solution were added and the mixture was incubated at 37°C for 1 hour. The solution was used for enzyme digestion and further cloning.

[0197] 2. Large scale plasmid extraction

[0198] 500ml bacterial solution was taken and centrifuged at 5000rpm for 10min at 4°C. The bacterial cells were collected by centrifugation after washing with 100ml STE. Solution I (50mM Glucose, 25mM Tris-HCl, 10mM EDTA, pH 8.0) 18ml and lysozyme (10mg / ml, 1% SDS) 2ml were added and the mixture was incubated at room temperature for 10min. Solution II (0.2N NaOH, 1% SDS) 40ml was added and the mixture was gently shaken until the bacterial solution became transparent. The mixture was incubated in an ice bath for 5min. Solution III (3M NaAc, pH 4.8) 60ml was added and the mixture was gently shaken and centrifuged at 5000rpm for 10min. The supernatant was carefully removed and added to 2 times volume of pre-cooled absolute ethanol in an ice bath at -20°C for 2 hours. The mixture was centrifuged at 5000rpm for 15min. The ethanol was removed and 500ml 70% ethanol was added to wash away the salt. After centrifugation, 50ml TE was added and the mixture was incubated at 37°C for 1 hour. The solution was used for enzyme digestion and further cloning.

[0199] 4.8) 20 ml, mix well and then 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 then place at -20 °C overnight, centrifuge at 12,000 rpm for 15 min, discard the supernatant, and then 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 then incubate at 37 °C for 30 min, and then place on a Sepharose 2B column (1 X 10 cm, equilibrated with TE), and then collect the first peak (large molecular weight DNA) using TE (pH 7.6) as eluent. After adjusting the volume of the collected sample, add 2 volumes of pre-cooled absolute ethanol and 1 / 10 volume of 3 M NaAc (pH 5.2), mix well, place in a refrigerator at -20 °C overnight, centrifuge at 12,000 rpm for 15 min, discard the supernatant, wash the precipitate with 70% ethanol for 1-2 times, dry, dissolve in 1 ml of TE, and then 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 cryopreservation.

[0200] (V) Preparation of E. coli competent cells and transformation of recombinant plasmid

[0201] Inoculate the E. coli host cells in 2 ml of LB medium, and then shake overnight at 37 °C. The next day, inoculate 500 ml of the overnight culture in 50 ml of LB medium, and then shake at 37 °C at 300 rpm for 1 hour until the bacterial density reaches 0.5 OD600. Centrifuge at 5000 rpm for 5 min at 4 °C, discard the supernatant, suspend the bacterial cells in 25 ml of cold solution containing 100 mM CaCl2and 10 mM Tris-HCl (pH 7.4), and then place 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 bacterial cells in 2 ml of calcium chloride cold solution, and then place in an ice bath for 40 min. The bacterial cells can then be used for transformation.

[0202] 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 then place in an ice bath for 30 min. Then, heat shock at 42 °C for 2 min, add 500 μl of LB medium, and then 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 then mix the bacterial suspension gently using a gun tip. Divide the mixture into 50 μl and 150 μl, and then coat one LB agar plate containing 50 μg / ml of ampicillin with each, and then incubate in a 37 °C incubator for 8-16 hours.

[0203] The preparation method of the present embodiment for efficiently obtaining high-purity recombinant human serum albumin comprises the following steps:

[0204] 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;

[0205] S2, fermentation;

[0206] S3, purification.

[0207] 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.

[0208] The fermentation of step S2 specifically includes

[0209] S210, configuring a culture medium;

[0210] S220, electrode calibration;

[0211] S230, feeding;

[0212] S240, inoculation, inoculating the new host strain with high expression of human serum albumin;

[0213] S250, fermentation;

[0214] S260, tank release.

[0215] The recombinant expression vector comprises:

[0216] (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;

[0217] (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;

[0218] (3) a 3' termination sequence from the methylotrophic yeast, 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.

[0219] 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 so far in the world.

[0220] 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 DataBank, 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 multi-aspect auxiliary analysis, and finally the optimized gene coding human serum albumin is obtained.

[0221] The present application also relates to the optimized gene coding human serum albumin, which comprises the following components:

[0222] At least one marker gene available for screening in E. coli;

[0223] A DNA fragment of a replication origin capable of replicating in the E. coli host bacteria;

[0224] At least two marker genes available for screening in yeast.

[0225] In the optimized gene coding human serum albumin, the preferred codon of Pichia pastoris alcohol oxidase (AOX1) gene is selected and the preferred codon accounts for 90% of the total number of codons of the coding optimized gene.

[0226] 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, which is beneficial to the splicing, cloning and assembly of the synthesized gene.

[0227] 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).

[0228] The optimized gene encoding human serum albumin further comprises an expression reading frame of

[0229] 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;

[0230] A 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; 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 a 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, 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.

[0231] The chemically fully synthesized mature gene (1755bp) 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 lane 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 fully synthesized mature gene (1755bp) encoding human serum albumin is cut from the pUC18 cloning vector by EcoRI-NotI double digestion, and the corresponding size gel strip is cut off, and the fragment of the target gene is recovered by 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 HSA EcoRI-NotI fragment), and the specific recombinant vector flow chart is shown in Fig. 4, and the connection reaction system is:

[0232] The ligation was performed overnight at 16°C in a 20 μl reaction system.

[0233] 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.

[0234] 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:

[0235] a. Preparation of culture medium

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 4. 1M, pH 6.0 potassium phosphate buffer: 132 mL 1M K2HPO4, 868 mL 1M KH2PO4, pH 6.0, autoclaved, stored at 4°C.

[0240] 5. 10x YNB: YNB 134 g (with ammonium sulfate) dissolved in 1000 mL deionized water, filter sterilized, stored at 4°C.

[0241] 6. 500x B: 20 mg Biotin dissolved in 100 mL deionized water, filter sterilized, stored at 4°C.

[0242] 7. 10x M: 5 mL methanol mixed with 95 mL deionized water, filter sterilized, stored at 4°C.

[0243] 8. 10x GY: 100 mL glycerol mixed with 900 mL deionized water, autoclaved, stored at 4°C.

[0244] 9. 10x D: 100 g glucose dissolved in 1000 mL deionized water, filter sterilized, stored at 4°C.

[0245] 10. 1M sorbitol: 18.2 g D-sorbitol dissolved in 100 mL deionized water, filter sterilized, stored at 4°C

[0246] b. Cell preparation

[0247] 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;

[0248] 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;

[0249] 3. Centrifuge the cell culture at 4°C, 1500 g for 5 min, resuspend the cell pellet with 500 mL ice-precooled sterile water;

[0250] 4. Centrifuge as in step 3, resuspend the cell pellet with 250 mL ice-precooled sterile water;

[0251] 5. Centrifuge as in step 3, resuspend the cell pellet with 20 mL ice-precooled 1M sorbitol solution;

[0252] 6. Centrifuge as in step 3, resuspend the cell pellet with 1 mL ice-precooled 1M sorbitol solution, the final volume is about 1.5 mL;

[0253] Note: The 80 μl aliquots can be frozen, but this will affect the transformation efficiency (within 2 weeks).

[0254] c. Electroporation

[0255] 1. Add 80 μL of prepared Pichia pastoris competent cells and 5-20 μg of 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);

[0256] 2. Chill the transformation cuvette containing the transformation mixture for 5 min;

[0257] 3. According to the information provided by the electroporator, refer to other literature and multiple attempts, determine the appropriate voltage, current, capacitance and other parameters, according to the optimized parameters, carry out electroporation; the preferred parameters of the present application are: voltage 1500-1800 V; capacitance 25 μF; resistance 200-400 Ω, shock time 4-10 msec.

[0258] 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;

[0259] 5. Incubate at 28°C on a shaker for 1-2 h;

[0260] 6. Take 50-200 μL of Pichia pastoris host strain GS115 or CBS7435 transformation solution and spread on MD plates;

[0261] 7. Incubate in a 28°C incubator for about 3-4 days until many single colonies containing recombinant plasmid positive colonies (yeast transformants) appear.

[0262] d. Screening of multi-copy transformants

[0263] 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 transfer the bacterial solution to a centrifuge tube;

[0264] 2. Spread the obtained bacterial solution on YPD plates containing G418 resistance, 100 μL of bacterial solution per plate; 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.

[0265] 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 different copy number of Pichia pastoris transformants).

[0266] 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.

[0267] Table 1 Experimental results of the host bacteria GS115 by the electric transformation Note: In Table 1, the G418 resistance of 0.25 represents that the host bacteria contains one copy of the exogenous gene, and so on.

[0268] Table 2 Experimental results of the host bacteria CBS7435 by the electric transformation Note: In Table 2, the G418 resistance of 0.25 represents that the host bacteria contains one copy of the exogenous gene, and so on.

[0269] 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. The high expression clones are identified. Then, the high expression clones screened are preserved at low temperature by 15% glycerol, and used as the engineering strains for further expression test.

[0270] The pilot test induction experiment is carried out for the above engineering high expression strain. If it is the Pichia pastoris GS115 strain, the experimental scheme is as follows: the glycerol bacteria are inoculated in YPD liquid medium for rejuvenation, 1 ml of seed 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 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 cell density of the Pichia pastoris engineering strain generally reaches 18-20 OD600 units at this time. 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.

[0271] 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.

[0272] This example is a small-scale induction experiment on some Pichia pastoris GS115 or CBS7435 strains. Some results are shown in Figures 11, 12 and Figures 13, 14.

[0273] The engineered strains with obvious expression bands in the above-mentioned small-scale induction experiments were further subjected to high-cell-density fermentation experiments in 5L, 30L, 50L, and 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:

[0274] S210, preparation of culture medium;

[0275] The fermentation medium of this example was prepared as shown in Table 3 below.

[0276] Table 3 Preparation of fermentation medium (prepared according to 6L volume)

[0277] 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.

[0278] S220, electrode calibration, including pH electrode calibration and dissolved oxygen electrode calibration.

[0279] pH electrode calibration: first prepare the standard pH electrode solution of pH 6.86 and 4.00, wash bottle and paper towel. Connect the pH electrode to the electrode line of the fermenter, rinse the electrode with the wash 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 the electrode with the wash bottle, then absorb the water with the paper towel, insert the electrode into the pH 4.00 electrode solution, and similarly when the pH value displayed 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.

[0280] 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 to 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.

[0281] S230, feeding.

[0282] 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.

[0283] 100% analytical pure methanol 3L;

[0284] 50% glycerol feed 800ml:

[0285] 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.

[0286] S240, inoculation, inoculate the new host strain with high expression of human serum albumin;

[0287] Pichia pastoris engineering strain fermentation experiment process

[0288] 1. Seed preparation:

[0289] 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.

[0290] Inoculate the seed bacteria into the fermenter:

[0291] 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 ring inoculation. Flame ring 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 exhaust 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 ring, and then pour the seed bacteria and PTM1 into the tube. Do not mix the seed bacteria and PTM1 before inoculation.

[0292] Recombinant human serum albumin fermentation culture stage:

[0293] (1) Engineering bacteria basic culture stage

[0294] Start SCADA process control software for data recording, fermentation time recording and batch number recording, and open atlas recording when engineering bacteria fermentation starts. Record 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. Control parameters are set as follows: control pH at 5.5±0.1 with ammonia, and control temperature at 28±1°C; control DO value above 30, air flow at 2vvm, and 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 no operation is performed on the fermenter ventilation, stirring, and tank pressure), enter the transition culture phase; take samples to detect OD 600 Should be around 50.

[0295] (2) Engineering bacteria transition culture stage:

[0296] The transition culture phase of the engineered bacteria is used to further increase the biomass of the engineered bacteria. Fermentation data is recorded hourly. During the transition culture phase, 50% glycerol needs to be fed in: after the DO rebounds, immediately feed in 50% glycerol (approximately 10 ml of PTM1 solution containing biotin at 12 ml / L); simultaneously, adjust the stirring speed to the maximum specified speed for the fermenter, the air flow rate to 2 vvm, and the tank pressure to 0.04–0.08 MPa; under these conditions, the feed rate should be controlled to maintain the DO value at 30 ± 10. DO-Spike should be performed at least once every hour, ensuring the DO-Spike response time is below 60 seconds. The feeding cycle is approximately 4–5 hours; when feeding glycerol, the pH setpoint is adjusted every hour to ensure that the pH reaches the required induction pH value after the glycerol feeding is completed (in this fermentation, the pH during the induction phase is the same as the pH during the culture phase, both being 5.7, therefore no pH adjustment is needed); at the end of the transition culture phase, OD... 600 The concentration is approximately 100. At this point, a sample is taken to measure the wet weight of the bacterial culture and retained for 0 hours. Thereafter, the wet weight is measured for each subsequent sample, but the OD (octane rating) is not measured. Glycerol feeding is stopped, and the methanol induction phase begins.

[0297] (3) Engineered bacteria induction and expression stage:

[0298] After stopping the addition of glycerol and observing a rebound in dissolved oxygen (DO), prepare to add methanol (add 12 ml / L of PTM1 to the methanol); add 4 g / L of methanol at once (in this experiment, 24 g of methanol was added at once, or 30 ml of methanol can be added through the feeding gasket using a disposable syringe). After the added methanol is depleted (DO rebound), start adding methanol via DO-Spike. A DO-Spike response time of less than 60 seconds is ideal, keeping the DO value between 10-30. Simultaneously with methanol addition, adjust the temperature to the project-specified temperature (24℃ in this experiment). Control the pH at the project-spike specified pH 5.7. During the engineered bacteria induction phase, perform DO-Spike every 12 hours and record the response time and other fermentation data. Sample every 12 hours to check pH and engineered bacteria wet weight, centrifuge, and add 100 μl of the supernatant to the electrophoresis buffer. Boil at 100℃ for 5 minutes, then store at -20℃. Store the remaining supernatant at 2-8℃ for later testing or electrophoresis analysis. Label the samples carefully during sampling. Throughout the entire Pichia pastoris engineered bacteria induction phase, maintain the methanol concentration in the culture medium within the tank between 0.2-0.9%.

[0299] (4) Place the can

[0300] The Pichia pastoris engineered strain was used in a fermentation tank experiment. The induction time for expression was approximately 200 hours, followed by tank discharge, solid-liquid separation, and subsequent processing. The supernatant was collected for subsequent purification.

[0301] The detection of the target protein in the fermentation test process of the Pichia pastoris engineering bacteria is as follows:

[0302] 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.

[0303] 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

[0304] 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

[0305] Table 6 Experimental results of producing gene recombinant human serum albumin by high-density fermentation technology

[0306] In Table 6, C represents the CBS7435 engineering bacteria, and G represents the GS115 engineering bacteria

[0307] The step S300 is specifically as follows:

[0308] S310, taking out the fermentation broth;

[0309] S320, centrifuging the fermentation broth;

[0310] S330, membrane filtering the supernatant;

[0311] S340, heat treating;

[0312] S350, secondary membrane filtering;

[0313] S360, column chromatography;

[0314] The step S310 specifically comprises taking the fermentation broth from the fermenter after the fermentation is completed in step S2.

[0315] The step S320 specifically comprises centrifuging the fermentation broth taken in step S310 at 9000 rpm, with temperature control below 30℃.

[0316] The step S330 specifically comprises filtering the fermentation supernatant after centrifugation in step S320 by hollow fiber membrane to obtain the filtered fermentation supernatant.

[0317] 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.

[0318] The step S350 specifically comprises filtering the heat-treated fermentation supernatant obtained in step S340 again by hollow fiber membrane to obtain the fermentation supernatant to be purified.

[0319] 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.

[0320] The column chromatography is selected from

[0321] TH-MC composite mode SP cation exchange column chromatography and hydroxyapatite type II (HAP II) chromatography and DEAE anion exchange column chromatography, etc.

[0322] or

[0323] TH-MC composite mode SP cation exchange column chromatography and hydroxyapatite type II (HAP II) column chromatography and TA-phenyl-HIC hydrophobic column chromatography, etc.

[0324] 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, which shows a single peak, and the results are shown in FIG. 20.

[0325] 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 the mass spectrometry-SEC, RP elution condition under non-reducing condition and reducing condition were compared and researched, and the results showed that the total ion chromatogram (TIC) chromatogram under the above conditions showed a single peak, and the peak time and peak type showed high consistency (see Figures 21 and 22). Further experiments, the molecular weight was determined by electrospray mass spectrometry method, and the results showed that the molecular weight of the recombinant human serum albumin sample (No. P7901-PH-E) provided by our company and the rHSA national standard sample was 66477.0 and 66487.5 Da respectively, which showed high consistency with the theoretical molecular weight of HSA 66472 Da, see Figure 23.

[0326] The construction method of the new host bacteria of the high expression of human serum albumin in the embodiment has the following beneficial effects:

[0327] 1) The purpose gene is obtained by molecular level whole sequence optimization design, so that the Pichia pastoris is most suitable for expression; 2) At the cell level, the latest Pichia pastoris expression system host bacteria CBS7435 is introduced for gene expression of human serum albumin; the G418 resistance gene is used to screen the Pichia pastoris transformant containing high copy number of exogenous genes, which is used to construct the high-level expression of recombinant human serum albumin Pichia pastoris engineering bacteria; 3) At the engineering level, the expression level of the target protein is further improved by exploring the optimization scheme of the fermentation culture process and the reactor process control; 4) On the purification process, the serious defects existing in the existing purification process of recombinant human serum albumin are analyzed and learned, and the latest purification medium, purification technology and purification method at home and abroad are introduced, only the fermentation broth solid-liquid separation, heat treatment and ultrafiltration concentration are used for pretreatment, and then the whole purification process is completed through three-step column chromatography, and the purity of the recombinant human serum albumin reaches more than 99%, and the recovery rate of the whole purification process reaches more than 60%.

[0328] The above embodiments only express certain embodiments of the present application, which are described in detail, but cannot be understood as a limitation on the scope of the present application; it should be pointed out 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 belong to the protection scope of the present application; therefore, the protection scope of the present application patent should be subject to the appended claims.

[0329] The sequence involved in the present application is:

Claims

1. A method for constructing a novel host bacterium that highly expresses human serum albumin, characterized in that: The construction method comprises S100, designing an optimized gene coding human serum albumin; S200, constructing a recombinant expression vector; S300, transforming the recombinant expression vector obtained in step S200 into a Pichia pastoris host strain by an electrotransformation method to obtain the novel host strain.

2. The method for constructing a novel host bacterium for high expression of human serum albumin according to claim 1, characterized in that: The nucleotide sequence contained in the optimized gene coding human serum albumin in step S100 is shown in SEQ ID NO: 2, and the amino acid sequence contained therein is shown in SEQ ID NO:

3.

3. The method for constructing a novel host bacterium for high expression of human serum albumin according to claim 1, characterized in that: In the optimized gene coding human serum albumin in step S100, the biased codons of the Pichia pastoris alcohol oxidase (AOX1) gene are selected and controlled to account for 90% of the total number of codons of the optimized gene.

4. The method for constructing a novel host bacterium for high expression of human serum albumin according to claim 1, characterized in that: In the optimized gene coding human serum albumin in step S100, 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.

5. The method for constructing a novel host bacterium for high expression of human serum albumin according to claim 1, characterized in that: In the optimized gene coding human serum albumin in step S100, 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%.

6. The method for constructing a novel host bacterium for high expression of human serum albumin according to claim 1, characterized in that: In the optimized gene coding human serum albumin in step S100, the expression reading frame comprises the insertion of a restriction enzyme BamHI site at the 5' end into the 5' regulatory region (promoter region) of the AOX1 gene, followed by a CCAAACGATG 10-deoxy oligonucleotide (containing the Kozak sequence of eukaryotic genes, i.e. AXXATG), followed by a Saccharomyces cerevisiae-derived a-mating pheromone leader peptide (85 amino acids) sequence, and then the HSA mature gene is inserted between EcoRI and NotI of the multiple cloning site of the pPIC9K recombinant expression vector. A 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; a 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.

7. The method for constructing a novel host bacterium highly expressing human serum albumin according to any one of claims 1-6, characterized in that: In step S200, the recombinant expression vector is constructed by The chemically fully synthesized optimized gene coding human serum albumin is cut off from the cloning vector by EcoRI-NotI double digestion, and the corresponding size gel strip is cut off, and the target gene fragment is recovered by DNA recovery Kit; the fragment containing the target gene is cloned into the recombinant expression vector subjected to corresponding double digestion, so as to construct the recombinant expression vector.

8. The method for constructing a novel host bacterium for high expression of human serum albumin according to claim 7, characterized in that: In step S300, the recombinant expression vector obtained in step S200 is linearized by using restriction enzymes SacI or Bgl II, and the linearized recombinant expression vector is transformed into a Pichia pastoris host strain by an electrotransformation method to obtain the novel host strain.

9. The method for constructing a novel host bacterium for high expression of human serum albumin according to claim 8, characterized in that: The nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO:

1.

10. The method for constructing a novel host bacterium for high expression of human serum albumin according to claim 7, characterized in that: The recombinant expression vector is any one of pPIC9, pPIC3, pPICZalphaABC, pPIC3.5K, pHIL-S1, pHIL-D2, pA0804, pA0815, pGAPZalphaABC, pPIC6alphaABC, pPIC9K.

11. The method for constructing a novel host bacterium for high expression of human serum albumin according to claim 10, characterized in that: The recombinant expression vector is HSA-pPIC9K.

12. The method for constructing a novel host bacterium for high expression of human serum albumin according to claim 8, characterized in that: The Pichia pastoris host strain is Pichia pastoris CBS7435.

13. The method for constructing a novel host bacterium for high expression of human serum albumin according to claim 1, characterized in that: The new host strain 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.

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