A method for preparing prostate-specific membrane antigen

Abstract

The present invention provides a method for preparing prostate-specific membrane antigen. Specifically, the present invention provides a nucleotide sequence of prostate-specific membrane antigen optimized by codons. Transferring this nucleotide sequence into cells for expression can significantly improve the yield of prostate-specific membrane antigen.

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 develops in the prostate gland and is the most common malignancy of the male genitourinary system. Prostate cancer is a particularly slow-growing cancer that is difficult to detect in its early stages. Treatment options include radical prostatectomy and surgery, or medical castration.

[0003] Prostate-specific membrane antigen (PSMA) is a multifunctional type II transmembrane protein present on the prostate cell membrane, composed 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 correlated with disease severity. Therefore, PSMA remains a highly sensitive and 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 from malignant prostate tissue. Its expression increases with advanced clinical stage, positively correlating with tumor malignancy. Therefore, PSMA is a more specific prostate cancer marker than PSA and can be used as an important indicator for prostate cancer prognosis.

[0005] Therefore, recombinantly expressed PSMA protein can serve as a target for drug screening, evaluating the inhibitory or activating effects of drugs on PSMA protein. It can also be used to prepare immunotherapeutic 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 like prostate cancer.

[0006] Therefore, developing an efficient method suitable for large-scale production of PSMA is of great significance to this field. 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 embodiment, the polynucleotide encodes the 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 further 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. 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 group consisting of 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 a human.

[0022] In another preferred embodiment, the host cell is selected from the group consisting of 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:

[0025] (a) culturing the host cell of the third aspect of the present invention under conditions suitable for culture, thereby obtaining a culture; and

[0026] (b) isolating and purifying prostate-specific membrane antigen protein from the culture.

[0027] In another preferred embodiment, in step (b), the process comprises:

[0028] (b1) The PSMA protein was isolated from the culture supernatant using nickel column affinity purification.

[0029] In another preferred embodiment, in step (b), the method further comprises:

[0030] (b2) Purifying the PSMA protein using a Superdex 200 pg chromatography column to obtain a highly pure PSMA protein.

[0031] In another preferred embodiment, the purification method includes: nickel column affinity purification.

[0032] In a preferred aspect, the present invention provides a method for preparing a prostate-specific membrane antigen protein, comprising the steps of:

[0033] (i) synthesizing a nucleic acid construct corresponding to a prostate-specific membrane antigen recombinant protein and constructing the nucleic acid construct into a eukaryotic expression vector to obtain a prostate-specific membrane antigen recombinant expression vector;

[0034] (ii) transfecting the prostate-specific membrane antigen recombinant expression vector into HEK293 cells using a transient expression system;

[0035] (iii) culturing the transfected HEK293 cells under suitable culture conditions to obtain a culture;

[0036] (iv) centrifuging the culture to collect the supernatant, and filtering and purifying to obtain the prostate-specific membrane antigen recombinant protein;

[0037] The nucleic acid construct sequence corresponding to the prostate-specific membrane antigen protein is shown in SEQ ID NO: 2.

[0038] In another preferred embodiment, the amino acid sequence of the prostate-specific membrane antigen recombinant protein is shown in SEQ ID NO: 5.

[0039] 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.

[0040] 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 to prepare a preparation or composition.

[0041] In another preferred embodiment, the preparation is a laboratory preparation.

[0042] In another preferred embodiment, the composition is a pharmaceutical composition.

[0043] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1A-Figure 1C An alignment of the original PSMA codons and the optimized codons selected according to the present invention is shown.

[0045] Figure 2 The figure shows the electrophoresis pattern of Ni column purification. The target protein was eluted at 20mM and 250mM imidazole concentrations with a purity of more than 85%; M: Marker; S: Cell secretion fluid reduction; FT: Ni column flow-through fraction reduction; F0: Buffer A wash fraction reduction; F114: Triton X-114 wash fraction reduction; W1: Buffer A wash fraction reduction; W2: Buffer B elution fraction reduction; E1-E15: Buffer C elution fractions and separate collection and reduction; R: Residual fraction on the column reduction; E2 (non-reduced): E2 fraction non-reduced electrophoresis.

[0046] Figure 3 The electrophoresis pattern of Ni column purification is shown. Here, S represents cell sap; FT represents Ni column flow-through; F0 represents Buffer A wash; M represents marker; 114 represents TX-114 wash; W1 represents Buffer A elution; W2 represents Buffer B elution fraction; E1-E6 represents Buffer C elution fractions; R represents column residue; and E3NR represents Buffer C elution fractions collected in E3 tube without reduction.

[0047] Figure 4 The electrophoresis pattern of Ni column purification is shown. Wherein, S: cell sap; FT: Ni column flow-through; E20: 20mM imidazole elution; E50: 50mM 1mi imidazole elution; E500: 500mM 1mi imidazole elution; R: column residue; NR: 500mM 1mi imidazole elution without reduction.

[0048] Figure 5 The electrophoresis pattern of Ni column purification is shown. Here, S represents cell sap; FT represents Ni column flowthrough; E20 represents 20 mM imidazole elution; E250 represents 250 mM 1 μm imidazole elution; NR represents 250 mM 1 μm imidazole elution without reduction; E500 represents 500 mM 1 μm imidazole elution; R represents column residue; and M represents marker.

[0049] Figure 6 The Superdex 200 pg chromatogram shows two peaks: the first peak with a retention volume of 78 ml is a high polymer, and the second peak with a retention volume of 102 ml is the main peak of the target protein.

[0050] Figure 7 The Superdex 200pg electrophoresis pattern is shown. The target protein in the main peak has a purity exceeding 95%. S: molecular sieve reduction before sample loading; M: marker; A3-A11 & B12-B3: molecular sieve elution fractions, which were collected and reduced separately.

[0051] Figure 8 The electrophoresis pattern of the quality control is shown. The purity is above 95%; M: Marker; R: final purified fraction.

[0052] Figure 9 The data actually measured for the proteins tested for activity in Example 6 are shown. DETAILED DESCRIPTION

[0053] After extensive and in-depth research and extensive screening, the present invention unexpectedly discovered a human prostate-specific membrane antigen (PSMA) nucleotide sequence with high production in human eukaryotic cells. The human PSMA coding sequence of the present invention is a PSMA nucleotide sequence obtained after optimizing various factors, including codon preference and mRNA secondary structure. The optimal human PSMA coding sequence of the present invention (SEQ ID No: 2) can significantly increase PSMA production (relative expression level of approximately 7 compared to the wild-type coding sequence). The recombinant PSMA protein of the present invention has modifications, such as glycosylation, that are almost identical to those of the native human PSMA protein and exhibits excellent solubility and biological activity. Based on this, the present invention was completed.

[0054] the term

[0055] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0056] As used herein, "isolated" or "isolated and purified" means that a substance has been separated from its original environment (in the case of a naturally occurring substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state within living cells are not isolated and purified. However, the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances with which they are naturally present.

[0057] Prostate-specific membrane antigen

[0058] 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 protein with glycosylation modification of the present invention," "polypeptide of the present invention," or "PSMA polypeptide of the present invention" are used interchangeably to refer to a PSMA extracellular domain protein (the extracellular domain of PSMA or the protein represented by amino acids 44-750 of the PSMA amino acid sequence) in a correctly glycosylated form expressed using human-derived eukaryotic host cells (somatic cells) and using the optimized sequence of the present invention.

[0059] Prostate-specific membrane antigen (PSMA) is a multifunctional type II transmembrane protein present in the prostate cell membrane, consisting of 750 amino acid residues. Amino acid residues 1-19 constitute the cytoplasmic portion; amino acid residues 20-43 form a helical structure, representing the transmembrane domain; and amino acid residues 44-750 constitute the extracellular portion of the protein, representing its primary functional structure. The amino acid sequence of PSMA from residues 44-750 is shown in SEQ ID NO: 5.

[0060] 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.

[0061] Optimization of PSMA coding sequence

[0062] In the present invention, the inventors optimized the coding sequence of PSMA by various means, 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 DNA GC content, optimizing gene translation start frame, optimizing translation termination sequence frame, etc.

[0063] There are 64 different genetic codes, but most organisms tend to use a subset of these codons. The most frequently used codons are called optimal codons, while those that are less frequently used are called rare or low-usage codons. Different species exhibit some degree of variation or preference in codon usage, which can affect the expression of recombinant proteins. Gene redesign, which utilizes preferred codons and avoids low-usage or rare codons, is called codon optimization.

[0064] In the present invention, human host cells (such as HEK cells, HEK293 cells or 293T cells) are used to express human proteins. The translation system of human host cells is already very compatible with the human wild-type coding sequence (or has been optimized through natural evolution). Therefore, when certain codons are replaced, the improvement effect is not obvious, or the protein yield is reduced instead of increased (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).

[0065] To this end, the applicants analyzed and optimized factors other than codon bias that may affect protein expression, including (but not limited to): eliminating sequences that destabilize mRNA, minimizing DNA secondary structure, adjusting DNA GC content, optimizing gene translation initiation and termination sequences, etc. The redesigned, multi-optimized PSMA coding sequence of the present invention can effectively remove or modify these unfavorable factors, thereby significantly increasing the expression of PSMA in human host cells, making exogenous protein production more efficient and economical.

[0066] Preparation and purification of PSMA protein

[0067] The PSMA proteins of the present invention (polypeptides of the present invention) are recombinant polypeptides produced from eukaryotic host cells (e.g., host cells derived from human somatic cells) using recombinant techniques. The polypeptides of the present invention are glycosylated, preferably with a human glycosylation pattern. Furthermore, the polypeptides of the present invention may or may not include an initial methionine residue.

[0068] Once the optimized coding sequence of the present invention is obtained, the sequence can be obtained in large quantities by recombinant methods, which usually involves cloning it into a vector, then transferring it into cells, and then isolating the sequence from the propagated host cells by conventional methods.

[0069] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0070] Methods using PCR technology to amplify DNA / RNA are preferably used to obtain the genes of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. Amplified DNA / RNA fragments can be separated and purified using conventional methods, such as by gel electrophoresis.

[0071] The present invention also relates to vectors comprising the polynucleotides of the present invention, host cells genetically engineered with the vectors or PSMA coding sequences of the present invention, and methods for producing the polypeptides of the present invention by recombinant technology.

[0072] 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:

[0073] (1) Transforming or transducing suitable host cells with the polynucleotide encoding human PSMA of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0074] (2) Host cells cultured in a suitable culture medium;

[0075] (3) Isolate and purify proteins from culture medium or cells.

[0076] 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, any plasmid or vector can be used as long as it can replicate and be stable in the host. A key feature of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0077] Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human PSMA and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter within the expression vector to direct mRNA synthesis. Representative examples of such promoters include the lac or trp promoters of Escherichia coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoter, retroviral LTRs, and other known promoters that 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.

[0078] In addition, the expression vector preferably contains 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.

[0079] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0080] 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 cells, COS cells, 293T cells and other animal cells.

[0081] When the polynucleotides of the present invention are expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting DNA factors, typically about 10 to 300 base pairs in length, that act on promoters to increase gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs on the late replication origin side), the polyoma enhancer on the late replication origin side, and adenovirus enhancers.

[0082] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0083] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. If desired, transformation can also be performed 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.

[0084] The obtained transformants can be cultured using conventional methods to express the PSMA polypeptides of the present invention. Depending on the host cells used, the culture medium used can be selected from various conventional culture media. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (e.g., temperature shift or chemical induction), and the cells are cultured for an additional period of time.

[0085] In the present invention, the PSMA recombinant protein of the present invention is secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods utilizing 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), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0086] In a preferred embodiment, the optimized coding sequence shown in SEQ ID No: 2 of the present invention can achieve a relative expression level of PSMA protein of approximately 688% under conditions of transient transfection into HEK293 cells compared to the wild-type coding sequence, and after isolation and purification, a PSMA protein concentration of approximately 0.87 mg / ml can be obtained.

[0087] The technical solution provided by the present invention has the following beneficial effects:

[0088] (a) The codon-optimized nucleotide sequence of the present invention can significantly increase the production of PSMA protein in human host cells (increased by about 6 times under transient transfection conditions).

[0089] (b) In the present invention, HEK293 and other human cells 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.

[0090] (c) In the present invention, when human cells such as HEK293 are used for production, the recombinant PSMA protein is secreted into the culture supernatant, which greatly simplifies the subsequent purification and helps to obtain high-purity (up to 99% or higher) and highly active PSMA protein.

[0091] (d) When codon bias alone was not sufficient to effectively increase expression, the present inventors analyzed and optimized factors other than codon bias that may affect protein expression, thereby significantly increasing the expression of PSMA in human host cells.

[0092] (e) Compared with existing codon-optimized recombinant proteins expressed in eukaryotic expression systems, the present invention has a significantly improved yield.

[0093] 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 invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally 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 by weight.

[0094] Example 1 Codon Optimization of Recombinant Protein Nucleotide Sequence and Construction of Recombinant Plasmid

[0095] 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.

[0096] The original nucleic acid sequence (or wild-type sequence or natural sequence) of PSMA is shown below:

[0097]

[0098] During sequence optimization, multiple optimizations (or multiple rounds of optimization) were performed, including (a) optimization based on codon bias, and (b) analysis and optimization of factors other than codon bias, including (but not limited to): elimination of sequences that destabilize mRNA, minimization of DNA secondary structure, adjustment of DNA GC content, optimization of gene translation start frame, optimization of translation termination sequence frame, etc., to eliminate factors that are unfavorable to the efficient expression of PSMA.

[0099] After optimization, dozens of optimized coding sequences were obtained. These coding sequences were artificially synthesized, vectors were constructed, and their expression performance was tested (see Example 2), from which coding sequences with excellent performance were selected.

[0100] The optimized coding sequences of the present invention include: 3 coding sequences shown in SEQ ID No: 2-4:

[0101] Optimized coding sequence 1:

[0102]

[0103] Optimized coding sequence 2:

[0104]

[0105] Optimized coding sequence 3:

[0106]

[0107] Among them, the optimized coding sequence 1 has a consistency of 74.16% with the original nucleotide sequence, and the sequence alignment is shown in the figure below. Figure 1A-Figure 1C shown.

[0108] The PSMA proteins encoded by the optimized coding sequences are identical to those encoded by the wt coding sequence (SEQ ID No: 1), and their amino acid sequences are shown in SEQ ID NO: 5:

[0109] KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQL AGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVH EIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYH LTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA (SEQ ID NO: 5)

[0110] Example 2 Expression of recombinant protein

[0111] The wild-type coding sequence (SEQ ID No: 1) and multiple optimized coding sequences (including SEQ ID NOs: 2-4) in Example 1 were respectively introduced into plasmid pCDNA3.4 (purchased from Suzhou Jinweizhi Biotechnology Co., Ltd.), and corresponding recombinant plasmids were constructed.

[0112] 2.1 Conversion

[0113] (1) Thaw DH5α competent cells at -80°C on ice and add 40 μL of sterile water to the plasmid powder to dissolve the plasmid;

[0114] (2) Gently blow the DH5α competent cells evenly;

[0115] (3) Add 2 μL of plasmid to DH5α competent cells;

[0116] (4) Place on ice for 30 min, activate at 42°C for 90 s, return to ice for 2-5 min, and add 100-500 μL of antibiotic-free LB medium;

[0117] (5) Shake at 37°C, 220 rpm for about 1 h until the OD value reaches 0.6-0.8;

[0118] (6) Take 100 μL of the transformed bacterial solution and evenly spread it on an LB plate containing Ampicilin antibiotics. Incubate the plate upside down at 37°C in a constant temperature incubator overnight.

[0119] 2.2 Plasmid extraction

[0120] 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 the A260 / A280 was 1.8.

[0121] 2.3 Recombinant protein expression

[0122] 2.3.1 Cell preparation before transfection

[0123] (1) Cell status detection: Take out cells (HEK293 cells) from the incubator and perform counting and viability detection.

[0124] Status description: Cells are in good condition (viability > 95%).

[0125] (2) Cell treatment: One day before transfection, dilute the cells to a density of approximately 1×10 6 / mL for transfection the next day.

[0126] 2.3.2 Transfection and expression

[0127] Prepare high-quality plasmids and cells in advance. The general transfection plasmid concentration is 1µg / mL, the plasmid and transfection reagent (PEI mass ratio is 1:3, the transfection buffer is KPM, and the transfection buffer system is 1 / 10 of the expression volume.

[0128] The specific steps are as follows:

[0129] (1) Ensure that the cell density for expression is approximately 2×10 6 cells / mL, cell viability as high as 95%

[0130] (2) 5 µg of plasmid and 15 µg of transfection reagent (PEI) were added to 250 µL of transfection buffer KPM. The transfection reagent and plasmid were mixed, allowed to stand at room temperature for 10 min, and then added to 5 mL of cells (gently invert to mix).

[0131] (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;

[0132] (4) After 7 days, the product was collected for small-scale purification.

[0133] Example 3 Purification of recombinant protein

[0134] 3.1 Sample preparation

[0135] Take the cell sample, centrifuge it at 6000 rpm for 10 min, and take the supernatant as S.

[0136] 3.2 Nickel column affinity purification

[0137] Purification was performed using the following buffers and steps:

[0138] BufferA: 20mM PB, 150mM NaCl, pH7.4

[0139] BufferB: 20mM PB, 150mM NaCl, pH7.4, 20mM Imidazole

[0140] BufferC:20mM PB, 150mM NaCl, pH7.4, 250mM Imidazole

[0141] (1) Take 5 mL of nickel column packing, add it to the gravity column, and flush the packing with 10 CV of 0.5% TritonX114;

[0142] (2) Wash the packing with 10CV water;

[0143] (3) Equilibrate the packing with 10CV of Buffer A;

[0144] (4) Load the sample onto the gravity column and recover the flow-through, which is recorded as Ft

[0145] (5) Use 10CV of Buffer A to re-equilibrate the packing, recorded as F0

[0146] (6) Wash the impurities with 10CV of Buffer A containing 0.1% Triton X114. Collect the wash solution for subsequent analysis and record it as F114.

[0147] (7) Use 10CV of Buffer A to wash away the residual TritonX114 in the filler, and collect the wash solution for subsequent analysis and record it as W1

[0148] (8) Elute with 10CV of Buffer B and collect the elution solution for subsequent analysis and record it as W2

[0149] (9) Elute with 10CV of Buffer C, approximately 4 mL per tube, and collect 15 tubes in total. Collect the eluate for subsequent analysis and record it as E1-E15

[0150] (10) Resuspend the filler with 1CV of Buffer C and draw 20µL as the column residue, which is recorded as R

[0151] (11) Wash the packing with 5CV of ultrapure water to remove the eluent from the packing;

[0152] (12) Wash the medium with 5CV of 20% ethanol and store at 4°C.

[0153] (13) Take 20µL of each sample (S, Ft, F0, F114, W1, W2, E1-15, R) from each step, add 5µL of 5× Loading Buffer and mix well, and incubate in a 95℃ metal bath for 5 minutes.

[0154] (14) Perform 10% SDS-PAGE analysis.

[0155] Experimental results

[0156] (a) The purification results using the wild-type coding sequence are as follows Figure 3 shown.

[0157] 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.

[0158] (b) The purification results using the optimized sequence of SEQ ID No: 2, as shown in FIG. Figure 2 shown.

[0159] 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 mixture was concentrated to 25 ml and subjected to SEC.

[0160] (c) The purification results using the optimized sequence of SEQ ID No: 3, as shown in FIG. Figure 4 shown.

[0161] 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.

[0162] (d) The purification results using the optimized sequence of SEQ ID No: 4, as shown in FIG. Figure 5 shown.

[0163] 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.

[0164] The purification results of four different coding sequences are summarized in Table 1 below:

[0165] Table 1

[0166]

[0167] 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-purified fraction of SEQ ID NO: 2 was selected for further size exclusion chromatography purification.

[0168] Example 4 Purification of PSMA Protein

[0169] In this example, the PSMA protein prepared in Example 3 was further purified by SEC to obtain a PSMA protein with higher purity.

[0170] Sample: Sample purified by Ni column prepared in Example 4 (concentrated to 5 mL)

[0171] Buffer:20mM MES, 150mM NaCl, pH5.5

[0172] Purification column: Superdex 200pg

[0173] (1) Treat the column with 0.5 M NaOH and H2O and then equilibrate with buffer for 1 CV until the UV280 and conductivity are stable.

[0174] (2) 1 mL / min sample loading;

[0175] (3) Elute 120 mL and collect 2 mL in each tube

[0176] (4) Check the SDS-PAGE test results.

[0177] result

[0178] Superdex200pg chromatogram Figure 6 This indicates that the PSMA protein has been effectively separated and purified, and the main peak shape indicates that the protein structure is uniform.

[0179] Superdex200pg chromatography electrophoresis pattern Figure 7 As shown. Combine B12-B6, totaling 11 mL, concentration: 0.8659 mg / ml; total mass: 9.524 mg. This indicates that the recombinant PSMA protein of the present invention has significantly improved yield and better solubility.

[0180] Example 5 Purity Detection

[0181] SDS-PAGE method: It is a denaturing polyacrylamide gel electrophoresis method; the principle of this method for separating proteins is based on

[0182] It is reported that most proteins can combine with the anionic surfactant sodium lauryl 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.

[0183] Test sample treatment: 6 μg of sample was pipetted and added with reducing test sample buffer at a ratio of 3:1, sample volume: reducing test sample buffer, mixed well, heated in a metal bath at 100°C for 5 minutes, cooled to room temperature, and loaded on SDS-PAGE.

[0184] Electrophoresis conditions: 12% SDS PAGE was used for electrophoresis at a constant voltage with an initial voltage of 80 V, which was adjusted to 200 V when entering the separation gel. Electrophoresis was stopped when bromophenol blue migrated to the bottom of the gel.

[0185] Fixation and staining (Coomassie Brilliant Blue method): Remove the electrophoresis gel slices and place them in the fixative for 60 minutes. Remove the film and place it in excess Coomassie Brilliant Blue staining solution for 1-2 hours. Discard the staining solution and place it in excess destaining solution. Change the destaining solution several times as needed. Destain until the gel background is transparent and then store it in the preservation solution.

[0186] Gel image processing: After the gel is developed, 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.

[0187] Molecular weight analysis: Calibrate with molecular weight standards. Purity analysis: Scan with a gel imager and calculate the results using peak area normalization.

[0188] The results are as follows Figure 8 As shown, where: M: Marker; R: final purified component.

[0189] The obtained PSMA protein had a molecular weight of 80.35 kDa and a purity of 99%. The theoretical molecular weight of PSMA is 70.5 kDa. Due to post-translational modifications such as glycosylation, the apparent molecular weight of the protein on electrophoresis is greater than the theoretical molecular weight. This indicates that the recombinant protein of the present invention possesses other modifications, such as glycosylation, compared to conventional PSMA proteins.

[0190] Example 6 Recombinant protein activity assay

[0191] Experimental Principle: This method uses N-acetyl-L-Asp-L-Glu as a substrate. The substrate is hydrolyzed by PSMA protein to produce L-Glu. After L-Glu is derivatized with OPA, fluorescence is measured at an excitation wavelength of 330 nm and an emission wavelength of 450 nm. The amount of N-acetyl-L-Asp-L-Glu hydrolyzed within a certain period of time (RFU) is quantitatively analyzed to reflect the activity of the PSMA protein.

[0192] 6.1 Reagent Preparation

[0193] (1) Assay buffer: 50 mM HEPES, 0.1 M NaCl, pH 7.5;

[0194] (2) Detected protein: PSMA;

[0195] (3) Substrate: N-acetyl-L-Asp-L-Glu, 10 mM dissolved in 40 mM NaOH;

[0196] (4) OPA: 50mg / mL stockin DMSO

[0197] (5) OPA buffer: 0.2M NaOH (containing 0.1% β-mercaptoethanol)

[0198] (6) Standard: L-Glu

[0199] 6.2 Experimental Procedure

[0200] (1) Preparation of standard curve: Dilute L-Glu (standard) to 10,000, 5,000, 2,500, 1,250, 625, 312.5, 156.25, and 0 nmol / L in assay 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 the excitation and emission wavelengths at 330 nm and 450 nm (topread) in endpoint mode.

[0201] (2) Dilute the protein to be tested to 0.4 μg / mL in assay buffer;

[0202] (3) Dilute the substrate to 40 μM in assay buffer;

[0203] (4) Experimental group: 125 μL of different concentrations of the test protein was mixed with 125 μL of 40 μM substrate. Blank group: 125 μL of different concentrations of the test protein was heated at 95°C for 5 min to inactivate, and then 125 μL of 40 μM substrate was added and mixed.

[0204] (5) Incubate at 37°C for 1 hour;

[0205] (6) The reaction was stopped by heating at 95°C for 5 minutes and then cooled to room temperature.

[0206] (7) Prepare a 15 mM OPA solution using OPA buffer.

[0207] (8) Add 250 μL of 15 mM OPA solution to all tubes and vortex.

[0208] (9) Incubate at room temperature for 10 minutes.

[0209] (10) Add 200 μL of the experimental group and blank group solutions to the ELISA plate.

[0210] (11) In endpoint mode, read at excitation and emission wavelengths of 330 nm and 450 nm, respectively.

[0211] 6.3 Formal Experimental Data Processing

[0212] 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. Figure 9 As shown, according to the parameters in Table 2 below, the activity is calculated as follows: 558.1 pmol / min / µg.

[0213] Table 2 Parameters for recombinant protein activity measurement

[0214]

[0215] discuss

[0216] Studies by the present inventors 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 the human wild-type sequence theoretically matches the preferred codons of human eukaryotic cells, the expression level of human PSMA is still very low.

[0217] To this end, the present inventors 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.

[0218] The results showed that the optimized nucleotide sequence (SEQ ID No: 2) of the present invention had a very low identity with the original nucleotide sequence (only about 74.16%).

[0219] 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.

[0220] In the present invention, when human cells such as HEK293 are used for production, the recombinant PSMA protein is secreted into the culture supernatant, making subsequent purification very simple. After separation and purification, the recombinant protein has a purity of 99% or higher and is highly soluble (concentration of approximately 0.87 mg / mL), achieving unexpected technical results.

[0221] The preparation of biologically active recombinant proteins is of great significance for both basic research and the production of biological preparations. Prior art CN114940710B discloses a method for expressing recombinant proteins in CHO cells. The present invention differs from this method in that it utilizes different expression cells and a different transformation method. The prior art examples provide a comparison of yields for different transformation methods, with electroporation yielding only 15 mg / L and PEI transfection yielding an even lower 0.2 mg / L. This indicates that the present invention (0.8659 mg / mL = 865.9 mg / L) achieves a significantly improved yield compared to existing codon-optimized recombinant proteins (15 mg / L) expressed in the same eukaryotic expression system.

[0222] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

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; the host cell is a HEK293 cell.

5. 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.

6. The method according to claim 5, characterized in that In step (b), comprising: (b1) The PSMA protein was isolated from the culture supernatant using a nickel column affinity purification method.

Images