Preparation method of prostate specific membrane antigen

Abstract

The invention provides a preparation method of a prostate specific membrane antigen. Specifically, the invention provides a codon optimized prostate specific membrane antigen nucleotide sequence, the nucleotide sequence is transferred into a cell for expression, and the yield of the prostate specific membrane antigen can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically, to a method for preparing prostate-specific membrane antigen. Background Art

[0002] Prostate cancer is an epithelial malignant tumor that occurs in the prostate gland and is the most common malignant tumor in the male urogenital system. Prostate cancer is a cancer that progresses particularly slowly and is difficult to detect in the early stages of the disease. It is mainly treated with radical prostatectomy and surgery or medical castration.

[0003] Prostate-specific membrane antigen (PSMA) is a multifunctional type II transmembrane protein present in the prostate cell membrane, consisting of 750 amino acid residues. PSMA has high prostate tissue specificity, with a specificity of up to 94.5% in distinguishing prostate cancer from other types of malignant tumors. Its expression level is closely related to the severity of the disease. Therefore, PSMA is still a very sensitive and highly specific antigen on prostate cancer cells and is considered an ideal therapeutic target for prostate cancer.

[0004] PSMA is superior to PSA (prostate-specific antigen) in distinguishing benign and malignant prostate tissues, and the later the clinical stage, the stronger its expression, which is positively correlated with the malignancy of the tumor. Therefore, PSMA is a more specific prostate cancer marker than PSA and can be used as an important indicator for the prognosis assessment of prostate cancer.

[0005] Therefore, the recombinantly expressed PSMA protein can be used as a target for drug screening to evaluate the inhibitory or activating effects of drugs on PSMA protein. It can also be used to prepare immunotherapy products such as specific antibodies and vaccines for the treatment and prevention of tumors. It can also be used to prepare diagnostic reagents, such as biomarkers for detecting diseases such as prostate cancer.

[0006] Therefore, developing an efficient method suitable for large-scale production of PSMA is of great significance to the art. Summary of the invention

[0007] The present invention provides a method that is efficient and suitable for large-scale production of PSMA.

[0008] In a first aspect of the present invention, an optimized polynucleotide encoding a prostate-specific membrane antigen (PSMA) recombinant protein is provided, the nucleotide sequence of the polynucleotide being shown in SEQ ID NO: 2.

[0009] In another preferred example, the polynucleotide encodes a PSMA protein having an amino acid sequence as shown in SEQ ID NO: 5.

[0010] In another preferred embodiment, the PSMA protein is human PSMA protein or a functional fragment thereof.

[0011] In the second aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide described in the first aspect of the present invention.

[0012] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0013] In another preferred embodiment, a protein tag is also inserted into the vector, such as a Flag tag, a Halo tag, a SNAP tag, a 6His tag, etc., preferably a 6His tag.

[0014] In another preferred embodiment, the protein tag is a 6his tag.

[0015] In another preferred embodiment, the vector is a eukaryotic cell expression vector.

[0016] In another preferred embodiment, the vector is a pCDNA3.4 vector.

[0017] In the third aspect of the present invention, a host cell is provided, wherein the host cell contains the vector described in the second aspect of the present invention, or the polynucleotide described in the first aspect of the present invention is integrated into its genome.

[0018] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0019] In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, yeast cells, and mammalian cells.

[0020] In another preferred embodiment, the host cell is a mammalian cell.

[0021] In another preferred embodiment, the host cell is a somatic cell derived from human.

[0022] In another preferred embodiment, the host cell is selected from the following group: HEK cells, HEK293 cells, 293T cells, Vero cells, or a combination thereof.

[0023] In another preferred embodiment, the host cell is HEK293 cell.

[0024] In a fourth aspect of the present invention, a method for preparing prostate-specific membrane antigen is provided, the method comprising the steps of: (a) culturing the host cell of the third aspect of the present invention under conditions suitable for culture, thereby obtaining a culture; and (b) isolating and purifying prostate-specific membrane antigen protein from the culture.

[0025] In another preferred embodiment, in step (b), comprising: (b1) The PSMA protein was isolated from the culture supernatant using a nickel column affinity purification method.

[0026] In another preferred embodiment, in step (b), the method further comprises: (b2) Purifying the PSMA protein using a Superdex 200 pg chromatography column to obtain a highly pure PSMA protein.

[0027] In another preferred embodiment, the purification method includes: nickel column affinity purification.

[0028] In a preferred aspect, the present invention provides a method for preparing a prostate-specific membrane antigen protein, comprising the steps of: (i) synthesizing a nucleic acid construct corresponding to a prostate-specific membrane antigen recombinant protein and constructing it into a eukaryotic expression vector to obtain a prostate-specific membrane antigen recombinant expression vector; (ii) transfecting the prostate-specific membrane antigen recombinant expression vector into HEK293 cells using a transient expression system; (iii) culturing the transfected HEK293 cells under suitable culture conditions to obtain a culture; (iv) centrifuging the culture to collect the supernatant, filtering and purifying to obtain the prostate-specific membrane antigen recombinant protein; The nucleic acid construct sequence corresponding to the prostate-specific membrane antigen protein is shown in SEQ ID NO: 2.

[0029] In another preferred example, the amino acid sequence of the prostate-specific membrane antigen recombinant protein is shown in SEQ ID NO:5.

[0030] In the fifth aspect of the present invention, a prostate-specific membrane antigen (PSMA) recombinant protein is provided, characterized in that the recombinant protein is produced by the cell described in the third aspect of the present invention, or is prepared by the method described in the fourth aspect of the present invention.

[0031] In the sixth aspect of the present invention, there is provided the use of the polynucleotide as described in the first aspect of the present invention, the expression vector as described in the second aspect of the present invention, the host cell as described in the third aspect of the present invention, or the recombinant protein as described in the fifth aspect of the present invention, characterized in that it is used for preparing a preparation or composition.

[0032] In another preferred embodiment, the preparation is a laboratory preparation.

[0033] In another preferred embodiment, the composition is a pharmaceutical composition.

[0034] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1A-Figure 1C An alignment of the original PSMA codons and the optimized codons selected according to the present invention is shown.

[0036] Figure 2 The Ni column purification electrophoresis spectrum is shown. The target protein is eluted at 20mM and 250mM imidazole concentrations, and the purity reaches more than 85%; M: Marker; S: cell secretion fluid reduction; FT: Ni column flow-through component reduction; F0: Buffer A washing component reduction; F114: Triton X-114 washing component reduction; W1: Buffer A washing component reduction; W2: Buffer B elution component reduction; E1-E15: Buffer C elution component and separate collection and reduction; R: column residual component reduction; E2 (non-reduction): E2 component non-reduction electrophoresis.

[0037] Figure 3 The electrophoresis spectrum of Ni column purification is shown. Among them, S: cell fluid; FT: Ni column flow-through; F0: Buffer A wash; M: Marker; 114: TX-114 wash; W1: Buffer A elution; W2: Buffer B elution fraction; E1-E6: Buffer C elution collection tube; R: column residue; E3NR: Buffer C elution collection E3 tube non-reduction.

[0038] Figure 4 The electrophoresis pattern of Ni column purification is shown, where S: cell sap; FT: Ni column flow-through; E20: 20mM imidazole elution; E50: 50mM Imi imidazole elution; E500: 500mM Imi imidazole elution; R: column residue; NR: 500mM Imi imidazole elution without reduction.

[0039] Figure 5 The electrophoresis spectrum of Ni column purification is shown. Among them, S: cell fluid; FT: Ni column flow through; E20: 20mM imidazole elution; E250: 250mM Imi imidazole elution; NR: 250mM Imi imidazole elution non-reduction; E500: 500mM Imi imidazole elution; R: column residue; M: Marker.

[0040] Figure 6 The Superdex200pg chromatogram shows two peaks, the first peak has a retention volume of 78ml, which is a high polymer, and the second peak has a retention volume of 102ml, which is the main peak of the target protein.

[0041] Figure 7 The Superdex200pg chromatography electrophoresis spectrum is shown. The purity of the target protein in the main peak reaches more than 95%; S: molecular sieve reduction before loading; M: Marker; A3-A11 & B12-B3: molecular sieve elution components and separate collection and reduction.

[0042] Figure 8 The electrophoresis pattern of quality control is shown. The purity is more than 95%; M: Marker; R: final purified component.

[0043] Fig. 9 The actual measured data of the protein whose activity was measured in Example 6 are shown. DETAILED DESCRIPTION

[0044] After extensive and in-depth research, the present invention unexpectedly discovered a human prostate-specific membrane antigen (PSMA) nucleotide sequence with high production performance in eukaryotic cells derived from humans through a large number of screenings. The human PSMA coding sequence of the present invention is a PSMA nucleotide sequence obtained after optimizing various factors such as codon preference and mRNA secondary structure. The optimal human PSMA coding sequence of the present invention (SEQ ID No: 2) can achieve a significant increase in PSMA production (relative expression level is about 7 compared to the wild-type coding sequence). The recombinant PSMA protein of the present invention has almost the same glycosylation and other modified forms as the natural human PSMA protein, and has excellent solubility and biological activity. On this basis, the present invention was completed.

[0045] the term As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0046] As used herein, "isolated" or "isolated and purified" means that a substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist with them in their natural state.

[0047] Prostate-specific membrane antigen As used herein, the terms "prostate-specific membrane antigen of the present invention", "PSMA protein of the present invention", "PSMA of the present invention", "PSMA of the present invention", "PSMA protein with glycosylation modification of the present invention", "polypeptide of the present invention", or "PSMA polypeptide of the present invention" are used interchangeably and refer to a PSMA extracellular domain protein (the extracellular domain of PSMA or the protein shown in amino acids 44-750 of the PSMA amino acid sequence) in a correct glycosylation form expressed by a eukaryotic host cell (somatic cell) derived from a human and using the optimized sequence of the present invention.

[0048] Prostate-specific membrane antigen (PSMA) is a multifunctional type II transmembrane protein present in the prostate cell membrane, consisting of 750 amino acid residues. Among them, amino acid residues 1-19 are present in the cytoplasm; amino acid residues 20-43 are a helical structure, belonging to the transmembrane part; amino acid residues 44-750 are the extracellular part of the protein, which is the main functional structural part of the protein. The amino acid sequence of PSMA at positions 44-750 is shown in SEQ ID NO: 5.

[0049] PSMA has high prostate tissue specificity, with a specificity of up to 94.5% in distinguishing prostate cancer from other types of malignant tumors. Its expression level is closely related to the severity of the disease. Therefore, PSMA remains a fairly sensitive and highly specific antigen on prostate cancer cells and is considered an ideal therapeutic target for prostate cancer.

[0050] Optimization of PSMA coding sequence In the present invention, the inventors optimized the coding sequence of PSMA by various different optimization methods, including but not limited to: redesigning and synthesizing the PSMA gene, such as eliminating rare codons, optimizing codon sequences, GC content, destabilizing sequences, minimizing DNA secondary structures, adjusting the GC content of DNA, optimizing the gene translation start frame, optimizing the translation termination sequence frame, etc.

[0051] There are 64 genetic codes, but most organisms tend to use a subset of these codons. Those that are used most frequently are called optimal codons, and those that are not used frequently are called rare or low-usage codons. Different biological species show some degree of difference or preference in codon usage, so the expression of recombinant proteins may be affected by codon usage. The redesign of genes by synthesizing genes using preferred codons and avoiding low-usage or rare codons is called codon optimization.

[0052] In the present invention, human host cells (such as HEK cells, HEK293 cells or 293T cells) are used to express human proteins, and the translation system of human host cells is already very compatible with the human wild-type coding sequence (or optimized by natural evolution). Therefore, when certain codons are replaced, the improvement effect will not be obvious, or the protein production will decrease instead of increase (such as the sequence of SEQ ID No: 4 causes the expression level to decrease). However, there is little room for codon optimization based on biological species (i.e., human cells).

[0053] To this end, the applicant has analyzed and optimized some other factors that may affect protein expression besides codon preference, including (but not limited to): eliminating sequences that destabilize mRNA, minimizing DNA secondary structure, adjusting DNA GC content, optimizing gene translation start frame, optimizing translation termination sequence frame, etc. The redesigned multi-optimized PSMA coding sequence of the present invention can efficiently remove or change these unfavorable factors, thereby significantly increasing the expression of PSMA in human host cells, making the production of exogenous proteins more efficient and economical.

[0054] Preparation and purification of PSMA protein The PSMA protein of the present invention (polypeptide of the present invention) is a recombinant polypeptide, which is produced from a eukaryotic host cell (e.g., a host cell derived from a human somatic cell) using recombinant technology. The polypeptide of the present invention is glycosylated, preferably the glycosylation is a human glycosylation pattern. In addition, the polypeptide of the present invention may or may not include an initial methionine residue.

[0055] Once the optimized coding sequence of the present invention is obtained, the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0056] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0057] The method of using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The DNA / RNA fragments amplified can be separated and purified by conventional methods such as by gel electrophoresis.

[0058] The present invention also relates to vectors comprising the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors of the present invention or the PSMA coding sequences, and methods for producing the polypeptides of the present invention by recombinant technology.

[0059] The polynucleotide sequences of the present invention can be used to express or produce recombinant PSMA polypeptides using conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally, the following steps are involved:

[0060] (1) Transforming or transducing a suitable host cell with a polynucleotide encoding human PSMA of the present invention, or with a recombinant expression vector containing the polynucleotide; (2) Host cells cultured in a suitable culture medium; (3) Isolate and purify proteins from culture medium or cells.

[0061] In the present invention, the human PSMA polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. In short, as long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0062] Methods well known to those skilled in the art can be used to construct expression vectors containing human PSMA encoding DNA sequences and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0063] In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance.

[0064] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0065] In the present invention, the host cell is a higher eukaryotic cell, such as a mammalian cell, especially a host cell derived from a human. Representative examples include: HEK cells, CHO, COS, 293T cells and other animal cells.

[0066] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0067] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0068] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0069] The obtained transformants can be cultured by conventional methods to express the PSMA polypeptide of the present invention. Depending on the host cells used, the culture medium used in the culture can be selected from various conventional culture media. The culture is carried out under conditions suitable for the growth of the host cells. When the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0070] In the present invention, the PSMA recombinant protein of the present invention is secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

[0071] In a preferred example, the optimized coding sequence shown in SEQ ID No: 2 of the present invention can achieve a relative expression level of PSMA protein of about 688% under the condition of transient transfection of HEK293 cells compared with the wild-type coding sequence, and a PSMA protein with a concentration of about 0.87 mg / ml can be obtained after separation and purification.

[0072] The technical solution provided by the present invention has the following beneficial effects: (a) The codon-optimized nucleotide sequence of the present invention can significantly increase the yield of PSMA protein in human host cells (increased by about 6 times under transient transfection conditions).

[0073] (b) In the present invention, human cells such as HEK293 are used for eukaryotic expression, and the glycosylation pattern of the prepared PSMA protein is more consistent with the glycosylation pattern of natural human PSMA.

[0074] (c) In the present invention, when human cells such as HEK293 are used for production, the recombinant PSMA protein will be secreted into the culture supernatant, which makes the subsequent purification very simple and helps to obtain high-purity (up to 99% or higher) and highly active PSMA protein.

[0075] (d) When the expression level could not be effectively increased based on codon preference alone, the inventors analyzed and optimized some other factors that may affect protein expression besides codon preference, thereby significantly increasing the expression level of PSMA in human host cells.

[0076] (e) Compared with the existing codon-optimized recombinant proteins expressed in the eukaryotic expression system, the present invention has a significantly improved yield.

[0077] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0078] Example 1 Codon optimization of recombinant protein nucleotide sequence and construction of recombinant plasmid In this example, based on the original nucleic acid sequence of PSMA (NC_000011.10), the coding sequence was optimized to achieve high-yield expression of PSMA protein.

[0079] The original nucleic acid sequence (or wild-type sequence or natural sequence) of PSMA is as follows: In sequence optimization, multiple optimizations (or multiple rounds of optimization) are performed, including (a) optimization based on codon preference, and (b) analysis and optimization of factors other than codon preference, including (but not limited to): eliminating sequences that destabilize mRNA, minimizing DNA secondary structure, adjusting the GC content of DNA, optimizing the gene translation start frame, optimizing the translation termination sequence frame, etc., to eliminate factors that are unfavorable to the efficient expression of PSMA.

[0080] After optimization, dozens of optimized coding sequences were obtained. For these coding sequences, vectors were constructed after artificial synthesis and their expression performance was tested (see Example 2), and coding sequences with excellent performance were selected.

[0081] The optimized coding sequences of the present invention include: 3 coding sequences shown in SEQ ID No: 2-4: Optimized coding sequence 1: Optimized coding sequence 2: Optimized coding sequence 3: Among them, the consistency between the optimized coding sequence 1 and the original nucleotide sequence is 74.16%, and the sequence alignment is shown in the figure below: Figure 1A-Figure 1C shown.

[0082] The PSMA proteins encoded by the above optimized coding sequences are the same as those encoded by the wt coding sequence (SEQ ID No: 1), and their amino acid sequences are shown in SEQ ID NO: 5: KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQL AGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVH EIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYH LTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA (SEQ ID NO: 5)

[0083] Example 2 Expression of recombinant protein The wild-type coding sequence (SEQ ID No: 1) and multiple optimized coding sequences (including SEQ ID NO: 2-4) in Example 1 were respectively introduced into plasmid pCDNA3.4 (purchased from Suzhou Jinweizhi Biotechnology Co., Ltd.), and recombinant plasmids were constructed accordingly.

[0084] 2.1 Conversion (1) Thaw DH5α competent cells from -80°C on ice, and add 40 μL of sterile water to the plasmid powder to dissolve the plasmid; (2) Gently blow the DH5α competent cells evenly; (3) Take 2 μL of plasmid and add it into DH5α competent cells; (4) Place on ice for 30 min, activate at 42°C for 90 s, place back on ice for 2-5 min, and add 100-500 μL of antibody-free LB medium; (5) Incubate the cells in a shaker at 37°C and 220 rpm for about 1 h until the OD value reaches between 0.6 and 0.8. (6) Take 100 μL of the transformed bacterial solution and spread it evenly on an LB plate containing Ampicilin antibiotics. Incubate the plate upside down in a 37°C constant temperature incubator overnight.

[0085] 2.2 Plasmid extraction The plasmid was extracted using the MN endotoxin-free plasmid extraction kit, and its concentration and A260 / A280 were determined using NanoDrop. The concentration was >200ug / ul, and A260 / A280 was 1.8.

[0086] 2.3 Recombinant protein expression 2.3.1 Cell preparation before transfection (1) Cell status detection: Take out cells (HEK293 cells) from the incubator and perform counting and viability detection.

[0087] Status description: The cells are in good condition (viability > 95%).

[0088] (2) Cell treatment: One day before transfection, dilute the cells to a density of approximately 1×10 6 / mL for transfection the next day.

[0089] 2.3.2 Transfection and expression Prepare high-quality plasmids and cells in advance. The general transfection plasmid concentration is 1µg / mL, the mass ratio of plasmid and transfection reagent (PEI is 1:3, the transfection buffer is KPM, and the transfection buffer system is 1 / 10 of the expression volume. The specific steps are as follows: (1) Ensure that the cell density for expression is approximately 2×10 6 / mL, cell viability as high as 95% (2) Add 5 µg of plasmid and 15 µg of transfection reagent (PEI) into 250 µL of transfection buffer KPM, mix the transfection reagent and plasmid, let stand at room temperature for 10 min, and then add to 5 mL of cells (gently invert to mix); (3) 24 h after transfection, add cell protein expression enhancer (293 0.6%) and 1× transient transfection nutrient supplement, and transfer to 32°C for culture; (4) After 7 days, the product was collected for small-scale purification.

[0090] Example 3 Purification of recombinant protein 3.1 Sample preparation Take the cell sample, centrifuge it at 6000 rpm for 10 min, and take the supernatant as S.

[0091] 3.2 Nickel column affinity purification The purification was performed using the following buffers and steps: BufferA: 20mM PB, 150mM NaCl, pH7.4 BufferB: 20mM PB, 150mM NaCl, pH7.4, 20mM Imidazole BufferC:20mM PB, 150mM NaCl, pH7.4, 250mM Imidazole (1) Take 5 mL of nickel column packing, add it to the gravity column, and rinse the packing with 10 CV of 0.5% TritonX114; (2) Wash the packing with 10CV water; (3) Use 10CV Buffer A to equilibrate the packing; (4) Load the sample onto the gravity column and recover the flow-through, recorded as Ft (5) Use 10CV Buffer A to re-equilibrate the packing, denoted as F0 (6) Wash with 10 CV of Buffer A containing 0.1% TritonX114 and collect the wash solution for subsequent analysis and record it as F114 (7) Use 10CV Buffer A to wash away the residual TritonX114 in the filler, collect the wash solution for subsequent analysis and record it as W1 (8) Elute with 10 CV of Buffer B and collect the elution solution for subsequent analysis and record it as W2 (9) Use 10CV Buffer C for elution, about 4 mL per tube, collect 15 tubes in total, collect the eluate for subsequent analysis and record it as E1-E15 (10) Resuspend the filler with 1CV of Buffer C and take 20µL as the column residue, recorded as R (11) Wash the packing with 5CV of ultrapure water to remove the eluent from the packing; (12) Wash the medium with 5 CV of 20% ethanol and store at 4°C. (13) Take 20 µL of each sample (S, Ft, F0, F114, W1, W2, E1-15, R) from each step, add 5 µL of 5× Loading Buffer, mix well, and place in a 95°C metal bath for 5 min (14) Perform 10% SDS-PAGE analysis.

[0092] Experimental Results (a) The purification results using the wild-type coding sequence are as follows Figure 3 shown.

[0093] Combine W2, E1-E6 to obtain a total of 80 ml, with a concentration of 0.1 mg / ml and a protein yield of 8 mg.

[0094] (b) The purification result using the optimized sequence of SEQ ID No: 2 is as follows: Figure 2 shown.

[0095] E1-E15 and W2 were combined to obtain a total of 110 ml with a concentration of 0.5 mg / ml and a protein yield of 55 mg. After combination, the fractions were concentrated to 25 ml and subjected to SEC.

[0096] (c) The purification result using the optimized sequence of SEQ ID No: 3, as shown in Figure 4 shown.

[0097] Combine E50 and E500 to obtain a total of 100 ml, with a concentration of 0.2 mg / ml and a protein yield of 20 mg.

[0098] (d) The purification result using the optimized sequence of SEQ ID No: 4 is as follows: Figure 5 shown.

[0099] Combine E250 and E500 to obtain a total of 100 ml, with a concentration of 0.1 mg / ml and a protein yield of 10 mg.

[0100] The purification results of four different coding sequences are summarized in Table 1 below: Table 1

[0101] Result analysis: Comparing the expression and Ni column purification results of SEQ ID NO: 1 (native sequence), codon-optimized sequences SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, the expression level of SEQ ID NO: 2 was significantly better than that of the native sequence and the other two codon-optimized sequences. Therefore, the Ni column purification component of SEQ ID NO: 2 was selected to continue the molecular exclusion chromatography purification.

[0102] Example 4 Purification of PSMA protein In this example, the PSMA protein prepared in Example 3 was further purified by SEC to obtain a PSMA protein with higher purity.

[0103] Sample: Sample purified by Ni column prepared in Example 4 (concentrated to 5 mL) Buffer:20mM MES, 150mM NaCl, pH5.5 Purification column: Superdex200pg (1) 0.5 M NaOH, H 2 After treating the column with O, equilibrate it with Buffer for 1CV until UV280 and conductivity are stable; (2) 1 mL / min sample loading; (3) Elute 120 mL and collect 2 mL in each tube (4) Check the SDS-PAGE test results.

[0104] result Superdex200pg chromatogram Figure 6 As shown, it indicates that the PSMA protein has been effectively separated and purified, and the main peak shape shows that the protein has good structural homogeneity.

[0105] Superdex200pg chromatography electrophoresis spectrum Figure 7 As shown. Combine B12-B6, a total of 11 mL, concentration: 0.8659 mg / ml; total mass: 9.524 mg. It can be seen that the recombinant PSMA protein of the present invention has a significantly improved yield and good solubility.

[0106] Example 5 Purity detection SDS-PAGE method: It is a denaturing polyacrylamide gel electrophoresis method. The principle of this method for separating proteins is based on It is reported that most proteins can combine with the anionic surfactant sodium dodecyl sulfate (by weight ratio) to form a complex, so that the negative charge carried by the protein molecule far exceeds the net charge of the natural protein molecule, eliminating the charge effect of different protein molecules and separating the proteins according to molecular size.

[0107] Test sample treatment: 6 μg of the sample was transferred and added with reduced test sample buffer at a ratio of 3:1. The sample volume was mixed and heated in a metal bath at 100°C for 5 minutes. The sample was cooled to room temperature and then loaded on SDS-PAGE.

[0108] Electrophoresis conditions: 12% SDS PAGE was used for electrophoresis, constant voltage electrophoresis, initial voltage was 80 V, adjusted to 200 V when entering the separation gel, and electrophoresis was stopped when bromophenol blue migrated to the bottom of the gel.

[0109] Fixation and staining (Coomassie Brilliant Blue method): Take out the electrophoresis gel slice, place it in the fixative for 60 minutes, take out the film and place it in excess Coomassie Brilliant Blue staining solution for 1-2 hours, discard the staining solution, place it in excess destaining solution, change the destaining solution several times as needed, and store it in the preservation solution after destaining until the gel background is transparent.

[0110] Gel image processing: After the gel color development process is completed, it is photographed or scanned. Usually, a commercial gel scanning system with data analysis software is used for photography and analysis to obtain molecular weight and purity information.

[0111] Molecular weight analysis: Calibrated according to molecular weight standards. Purity analysis: Scanned by gel imaging instrument, the results were calculated by peak area normalization method.

[0112] The results are as follows Figure 8 As shown, where: M: Marker; R: final purified component.

[0113] The molecular weight of the obtained PSMA protein is 80.35 KDa, and the purity is 99%. The theoretical molecular weight of the PSMA protein is 70.5 kDa. Due to post-translational modification processes such as glycosylation, the apparent molecular weight of the protein electrophoresis is greater than the theoretical molecular weight. It can be seen that the recombinant protein of the present invention has other modifications, such as glycosylation, compared with the general PSMA protein.

[0114] Example 6 Recombinant protein activity test Experimental principle: This method uses N-acetyl-L-Asp-L-Glu as a substrate. The substrate is hydrolyzed under the action of PSMA protein to produce L-Glu. After L-Glu is derivatized with OPA, fluorescence measurement is performed at an excitation wavelength of 330nm and an emission wavelength of 450nm. Quantitative analysis is performed based on the amount of N-acetyl-L-Asp-L-Glu hydrolyzed within a certain period of time (RFU) to reflect the activity of PSMA protein.

[0115] 6.1 Reagent preparation (1) Assay buffer: 50 mM HEPES, 0.1 M NaCl, pH 7.5; (2) Protein to be tested: PSMA; (3) Substrate: N-acetyl-L-Asp-L-Glu, 10 mM dissolved in 40 mM NaOH; (4) OPA: 50mg / mL stockin DMSO (5) OPA buffer: 0.2M NaOH (containing 0.1% β-mercaptoethanol) (6) Standard substance: L-Glu 6.2 Experimental procedures (1) Preparation of standard curve: Dilute L-Glu (standard) to 10000, 5000, 2500, 1250, 625, 312.5, 156.25, 0 nmol / L with detection buffer. Take 100 μL of each concentration gradient standard, add 100 μL of 15 mM OPA solution and vortex; incubate at room temperature for 10 minutes; add 200 μL of the solution to the ELISA plate and read at the excitation and emission wavelengths of 330 nm and 450 nm (topread) in endpoint mode.

[0116] (2) Dilute the protein to be tested to 0.4 μg / mL in assay buffer; (3) Dilute the substrate to 40 μM in assay buffer; (4) Experimental group: 125 μL of different concentrations of the test protein and 125 μL of 40 μM substrate were mixed. Blank group: 125 μL of different concentrations of the test protein were heated at 95°C for 5 min to inactivate, and then 125 μL of 40 μM substrate was added and mixed;

[0117] (5) Incubate at 37°C for 1 hour; (6) The reaction was stopped by heating at 95°C for 5 min and then cooled to room temperature.

[0118] (7) Prepare a 15 mM OPA solution using OPA buffer.

[0119] (8) Add 250 μL of 15 mM OPA solution to all tubes and vortex.

[0120] (9) Incubate at room temperature for 10 minutes.

[0121] (10) Add 200 μL of the experimental group and blank group solutions into the ELISA plate.

[0122] (11) In endpoint mode, read at excitation and emission wavelengths of 330 nm and 450 nm, respectively.

[0123] 6.3 Formal Experimental Data Processing Using GraphPad Prism 7 software, a standard curve was drawn with the average RFU value as the horizontal axis and the molar amount (pmol) of the standard amino acid L-glutamic acid as the vertical axis. Fig. 9 As shown, according to the parameters in Table 2 below, the activity = 558.1pmol / min / µg.

[0124] Table 2 Parameters for recombinant protein activity measurement

[0125] discuss The present inventors' studies have shown that when the wild-type coding sequence of human PSMA is used and human-derived eukaryotic cells are used as host cells for expression and production, although theoretically the human wild-type sequence matches the preferred codons of human eukaryotic cells, the expression level of human PSMA is still very low.

[0126] To this end, the inventors have analyzed and optimized some other factors that may affect protein expression besides codon preference, including (but not limited to): eliminating sequences that destabilize mRNA, minimizing DNA secondary structure, adjusting the GC content of DNA, optimizing the gene translation start frame, optimizing the translation termination sequence frame, etc.

[0127] The results showed that the optimized nucleotide sequence of the present invention (SEQ ID No: 2) had a very low consistency with the original nucleotide sequence (only about 74.16%).

[0128] However, introducing the optimized coding sequence with very low consistency into plasmid pCDNA3.4 and transiently transfecting HEK293 cells can significantly increase the production of PSMA protein in human host cells (increased by about 6 times), and the glycosylation pattern of the prepared PSMA protein is more consistent with the glycosylation pattern of natural human PSMA.

[0129] In the present invention, when human cells such as HEK293 are used for production, the recombinant PSMA protein will be secreted into the culture supernatant, which makes the subsequent purification very simple. After separation and purification, the purity of the recombinant protein reaches 99% or higher and the solubility is high (concentration is about 0.87 mg / mL), achieving unexpected technical effects.

[0130] The preparation of recombinant proteins with biological activity is of great significance for basic research and the production of biological preparations. The prior art CN114940710B has disclosed a method for expressing recombinant proteins in CHO cells. The difference between the present invention and the prior art CN114940710B is that the expression cells are different and the transformation methods are also different. In addition, the yield comparison of different transformation methods is given in the prior art embodiments, among which the yield of electrotransformation is only 15 mg / L, and the yield of PEI transfection is even lower, only 0.2 mg / L. It can be seen that the present invention (0.8659 mg / mL=865.9 mg / L) has a significantly improved yield relative to the existing codon-optimized recombinant protein (15 mg / L) expressed by the same eukaryotic expression system.

[0131] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An optimized polynucleotide encoding a prostate-specific membrane antigen (PSMA) recombinant protein, characterized in that: The nucleotide sequence of the polynucleotide is shown in SEQ ID NO:

2.

2. The polynucleotide according to claim 1, wherein The polynucleotide encodes a prostate-specific membrane antigen with an amino acid sequence as shown in SEQ ID NO:

5.

3. An expression vector, characterized in that: The expression vector contains the polynucleotide according to claim 1.

4. A host cell, characterized in that The host cell contains the vector according to claim 3, or the polynucleotide according to claim 1 is integrated into its genome.

5. The host cell according to claim 4, characterized in that The host cell is a mammalian cell.

6. The host cell according to claim 4, characterized in that The host cell is a somatic cell derived from human.

7. The host cell according to claim 4, characterized in that The host cell is selected from the group consisting of HEK cells, HEK293 cells, 293T cells, Vero cells, or a combination thereof.

8. The host cell according to claim 4, characterized in that The host cell is HEK293 cell.

9. A method for preparing prostate-specific membrane antigen protein, characterized in that: The method comprises the steps of: (a) culturing the host cell of claim 4 under conditions suitable for culture to obtain a culture; and (b) isolating and purifying prostate-specific membrane antigen protein from the culture.

10. The method according to claim 9, characterized in that In step (b), comprising: (b1) The PSMA protein was isolated from the culture supernatant using a nickel column affinity purification method.

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