CN114409800A - Method for preparing recombinant cystatin C - Google Patents

Method for preparing recombinant cystatin C Download PDF

Info

Publication number
CN114409800A
CN114409800A CN202111551776.9A CN202111551776A CN114409800A CN 114409800 A CN114409800 A CN 114409800A CN 202111551776 A CN202111551776 A CN 202111551776A CN 114409800 A CN114409800 A CN 114409800A
Authority
CN
China
Prior art keywords
protein
fusion protein
cys
polynucleotide
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202111551776.9A
Other languages
Chinese (zh)
Other versions
CN114409800B (en
Inventor
蒋析文
黄黉
颜青青
况修丽
肖兰花
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Da'an Gene Co ltd
Original Assignee
Guangzhou Da'an Gene Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Da'an Gene Co ltd filed Critical Guangzhou Da'an Gene Co ltd
Priority to CN202111551776.9A priority Critical patent/CN114409800B/en
Publication of CN114409800A publication Critical patent/CN114409800A/en
Application granted granted Critical
Publication of CN114409800B publication Critical patent/CN114409800B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a method for preparing recombinant cystatin C, and particularly relates to a method for preparing recombinant cystatin C by adopting genetic engineering escherichia coli to express Cys C protein in a recombinant fusion manner, wherein the expressed Cys C recombinant protein is fused with a specific tag sequence at the N end, so that the soluble high-efficiency expression in an escherichia coli system is realized, the protein stability is high, and the protein has excellent immunological activity.

Description

Method for preparing recombinant cystatin C
Technical Field
The invention belongs to the field of biotechnology. More particularly, it relates to a process for the preparation of recombinant cystatin C.
Background
Cystatin C (Cystatin C or Cys C for short) is also called Cystatin C, and is a cysteine protease inhibitor. It is an ideal endogenous marker reflecting Glomerular Filtration Rate (GFR) change, and cystatin C is used as a recognized renal function detection index at home and abroad at present.
The traditional method for extracting natural cystatin from tissues such as placenta, urine and the like has the problems of low efficiency, low purity, high purification cost and the like, has unknown potential risks, and has complex purification steps, so that the difficulty in obtaining high-purity protein is high, and the cross between polyclonal antiserum prepared by immunizing with the natural protein and other components in human serum is serious, thereby bringing great difficulty to the optimization of product performance. At present, the research on the expression of human Cys C by using an Escherichia coli expression system has been reported, but the expression of human Cys C in Escherichia coli belongs to heterologous expression, and the expression is reported to be in an inclusion body form. The expression product in the form of inclusion body must be subjected to complicated denaturation and renaturation treatments to recover the biological activity. Yeast and CHO cell system expression is also reported, but the production cost is high, the expression quantity is low, and the Cys C is not beneficial to the industrial production.
The preparation of cystatin C diagnostic kits requires strong specificity and high sensitivity anti-Cys C monoclonal antibodies, and high purity cystatin C protein must be obtained to obtain the monoclonal antibodies. Cys C has been extracted from patient urine, which is limited in source, high in cost and extremely low in yield. Therefore, the skilled in the art is dedicated to develop a preparation process of cystatin C protein with high expression efficiency and easy purification.
Disclosure of Invention
The invention provides a preparation process of cystatin C protein, which has high expression efficiency and is easy to purify.
In a first aspect of the present invention, there is provided a cystatin C fusion protein selected from the group consisting of:
(A) a polypeptide having an amino acid sequence shown in SEQ ID NO. 3;
(B) a polypeptide having a homology of not less than 90% (preferably, not less than 95% homology; etc., preferably, not less than 96% homology; most preferably, not less than 97% homology) with the amino acid sequence shown in SEQ ID NO. 3, and which retains the activity of the polypeptide shown in SEQ ID NO. 1;
(C) a derivative polypeptide which is formed by substituting, deleting or adding 1-5 amino acid residues of the amino acid sequence shown in SEQ ID NO. 3 and keeps the activity of the polypeptide shown in SEQ ID NO. 1.
In another preferred embodiment, the fusion protein is isolated.
In another preferred embodiment, the amino acid sequence of the cystatin C fusion protein is shown in SEQ ID NO 3.
In a second aspect of the invention, there is provided an isolated codon optimised polynucleotide encoding a fusion protein according to the first aspect of the invention.
In another preferred embodiment, the polynucleotide is selected from the group consisting of:
(a) polynucleotide with sequence shown in SEQ ID NO. 4;
(b) polynucleotide having a nucleotide sequence homology of 95% or more (preferably 98% or more) with the sequence shown in SEQ ID NO. 4;
(c) a polynucleotide complementary to any one of the polynucleotides of (a) - (b).
In a third aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the second aspect of the invention.
In another preferred embodiment, the expression vector is a pET-32a (+) expression vector.
In a fourth aspect of the invention, there is provided a host cell comprising an expression vector according to the third aspect of the invention or having integrated into its genome a polynucleotide according to the second aspect of the invention.
In another preferred embodiment, the host cell is a prokaryotic cell, preferably the host cell is e.coli, more preferably the Rosetta (DE3) strain of e.coli.
In a fifth aspect of the invention, there is provided a method of preparing a fusion protein according to the first aspect of the invention, comprising the steps of:
culturing the cell of the fourth aspect of the invention under conditions suitable for expression, thereby expressing the fusion protein of the first aspect of the invention; and isolating the fusion protein.
In a sixth aspect of the invention, there is provided a kit comprising a fusion protein according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention or an expression vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows that no Cys C protein soluble expression was seen in SB, TB, SOC media at 37 ℃ and 18 ℃.
FIG. 2 shows that the tag-conjugated Cys C protein is abundantly soluble in TB medium, i.e.in the supernatant.
FIG. 3 shows an electrophoretogram after purification of a large amount of expression product.
FIG. 4 shows the stability of recombinant fusion Cys C protein.
FIG. 5 shows that recombinantly expressed Cys C protein binds well to the antibody.
Detailed Description
The invention adopts genetic engineering escherichia coli to express the Cys C protein through recombinant fusion, and the expressed Cys C is fused with a specific tag sequence at the N end, so that the soluble high-efficiency expression in an escherichia coli system is realized, and the protein has high stability and immunological activity. The method for recombinant fusion expression of Cys C protein has the advantages of high yield, short production period, easy purification of the expression product, low cost and the like, and can realize the industrial production of Cys C protein.
Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.
Fusion proteins and their preparation
In the present invention, "fusion protein", "recombinant protein", "protein of the present invention", "fusion protein of the present invention" are used interchangeably and refer to a protein having the amino acid sequence shown in SEQ ID No. 3 or a sequence derived therefrom. The proteins of the invention may be monomers or multimers (e.g., dimers) formed from monomers. Furthermore, it is to be understood that the term also includes active fragments and derivatives of the fusion protein.
As used herein, "isolated" refers to a substance that is separated from its original environment (which, if it is a natural substance, is the natural environment). If the polynucleotide or polypeptide in the natural state in the living cell is not isolated or purified, but the same polynucleotide or polypeptide is isolated or purified if it is separated from other substances coexisting in the natural state.
As used herein, "isolated fusion protein" means that the fusion protein is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify the fusion protein using standard protein purification techniques. Substantially pure proteins produce a single major band on a non-reducing polyacrylamide gel.
The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.
The present invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded polypeptide.
As used herein, the term "primer" refers to a generic term for an oligonucleotide that, when paired with a template, is capable of synthesizing a DNA strand complementary to the template from its origin by the action of a DNA polymerase. The primer can be natural RNA, DNA, and any form of natural nucleotide. The primers may even be non-natural nucleotides such as LNA or ZNA etc. A primer is "substantially" (or "substantially") complementary to a particular sequence on one strand of the template. The primer must be sufficiently complementary to one strand of the template to begin extension, but the sequence of the primer need not be completely complementary to the sequence of the template. For example, a primer that is complementary to the template at its 3 'end and has a sequence that is not complementary to the template at its 5' end remains substantially complementary to the template. Primers that are not perfectly complementary can also form a primer-template complex with the template, so long as there is sufficient primer binding to the template, allowing amplification to occur.
The full-length nucleotide sequence or a fragment thereof of the fusion protein or an element thereof of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.
The invention also relates to vectors comprising the polynucleotides of the invention, as well as genetically engineered host cells encoded with the vector or fusion protein coding sequences of the invention, and methods for producing the proteins of the invention by recombinant techniques.
The polynucleotide sequences of the present invention may be used to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally, the following steps are performed:
(1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;
(2) a host cell cultured in a suitable medium;
(3) separating and purifying protein from culture medium or cell.
Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences encoding the proteins of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits 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 for E.coli.
Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.
The host cell may be a eukaryotic cell, such as a mammalian cell. Representative examples are: animal cells such as CHO, NS0, COS7, and 293 cells; prokaryotic cells, such as E.coli, are also possible.
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The protein obtained in the above method can be isolated and purified by various separation methods using its physical, chemical and other properties, if necessary. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
In a preferred embodiment of the invention, the amino acid sequence and gene sequence of Cys C according to the invention are as follows:
the base sequence:
MAGPLRAPLLLLAILAVALAVSPAAGSSPGKPPRLVGGPMDASVEEEGVRRALDFAVGEYNKASNDMYHSRALQVVRARKQIVAGVNYFLDVELGRTTCTKTQPNLDNCCFHDQPHLKRKAFCSFQIYAVPWQGTMTLSKSTCQDA(SEQ ID NO:1)
escherichia coli synonymous codon preference optimized base sequence:
ATGGCAGGACCCCTAAGGGCTCCATTATTGCTTCTGGCGATTTTGGCGGTAGCGCTGGCGGTGAGCCCGGCAGCAGGCTCGAGCCCAGGTAAACCGCCTCGCTTGGTCGGTGGTCCGATGGACGCCAGCGTTGAGGAAGAAGGCGTGCGTCGTGCGCTCGACTTCGCAGTTGGTGAATACAACAAAGCTTCTAATGATATGTATCATGGCCGTGCGCTGCAGGTTGTTCGCGCACGTAAGCAAATCGTGGCCGGTGTGAATTACTTCCTGGATGTTGAGTTGGGCCGTACGACCTGTACCAAGACCCAACCGAACCTGGACAACTGCCCGTTTCACGATCAGCCGCACCTGAAACGCAAGGCGTTTTGCTCCTTCCAAATCTATGCTGTGCCGTGGCAGGGCACCATGACTCTGAGCAAGTCCACCTGCCAGGACGCG(SEQ ID NO:2)。
in a preferred embodiment of the invention, the amino acid sequence and gene sequence of recombinant Cys C according to the invention are as follows:
the base sequence:
SSPGKPPRLVGGPMDASVEEEGVRRALDFAVGEYNKASNDMYHSRALQVVRARKQIVAGVNYFLDVELGRTTCTKTQPNLDNCCFHDQPHLKRKAFCSFQIYAVPWQGTMTLSKSTCQDASSPGKPPRLVGGPMDASVEEEGVRRALDFAVGEYNKASNDMYHSRALQVVRARKQIVAGVNYFLDVELGRTTCTKTQPNLDNCCFHDQPHLKRKAFCSFQIYAVPWQGTMTLSKSTCQDA(SEQ ID NO:3)
base sequence optimized by Escherichia coli synonymous codon preference
TCAAGTCCCGGAAAACCACCTAGGCTAGTAGGCGGTCCAATGGATGCCTCTGTTGAGGAAGAGGGCGTGCGTCGCGCGTTGGACTTTGCAGTTGGTGAATATAACAAAGCTTCCAACGATATGTACCATAGCCGTGCGCTGCAAGTAATGAGAGCTCGCAAGCAGATTGTCGCGGGTGTAAATTACTTCTTGGACGTTGAGCTCGGTCGTACCACCTGTACCAAGACCCAGCCGAATCTGGACAACTGCCCGTTTCACGATCAACCGCACCTGAAACGATAGGCGTTCTGCTCGTTCCAAATCTATGCAGTTCCGTGGCAGGGCACCATGACTCTGAGCAAAAGCACGTGCCAGGACGCGTCGAGCCCAGGTAAACCGCCTCGCTTGGTCGGTGGTCCGATGGACGCCAGCGTTGAGGAAGAAGGCGTGCGTCGTGCGCTCGACTTCGCAGTTGGTGAATACAACAAAGCTTCTAATGATATGTATCATGGCCGTGCGCTGCAGGTTGTTCGCGCACGTAAGCAAATCGTGGCCGGTGTGAATTACTTCCTGGATGTTGAGTTGGGCCGTACGACCTGTACCAAGACCCAACCGAACCTGGACAACTGCCCGTTTCACGATCAGCCGCACCTGAAACGCAAGGCGTTTTGCTCCTTCCAAATCTATGCTGTGCCGTGGCAGGGCACCATGACTCTGAGCAAGTCCACCTGCCAGGACGCG(SEQ ID NO:4)。
Genetically engineered cell
The invention provides a genetically engineered cell (host cell), which is a prokaryotic cell (preferably escherichia coli) and has an expression cassette of a fusion protein of the invention integrated in the genome of the cell; or the cell contains an expression vector containing an expression cassette for the fusion protein of the invention.
In a preferred embodiment, the expression cassette of the fusion protein of the invention comprises the following elements operably linked 5 'to 3': a promoter, an initiation codon, an ORF sequence of the fusion protein, and a stop codon.
In the present invention, the term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.
Materials and methods
1. Biological material
The host escherichia coli Rosetta (DE3) strain (purchased from tiangen biotechnology (beijing) limited), expression vector (purchased from kasey biotechnology limited), Cys C protein activity assay kit (Elisa kit, purchased from daley biotechnology limited, guangzhou) used in this experiment.
2. Vector construction
Carrying out optimization of the preference of synonymous codons of escherichia coli on the human Cys C gene and the recombinant fusion Cys C protein gene designed by the invention to obtain an optimized codon sequence, and constructing expression vectors after artificial synthesis, wherein the expression vectors are respectively connected to the vectors pET-28a (+) and pET-32a (+) (the downstream of an insertion site in the vector is provided with a coding (His). times.6 label gene).
3. Construction of host E.coli
Taking the expression vector, and transforming the expression vector into an Escherichia coli competent Rosetta (DE3) to obtain a host Escherichia coli.
4. Expression and purification of target genes
Fermenting and culturing the obtained host escherichia coli to obtain thalli, crushing the thalli, centrifuging, and performing Ni-column affinity chromatography on a bacterial liquid supernatant to obtain the human myoglobin target protein.
The invention has the beneficial effects that:
(1) the recombinant Cys C of the invention obviously enhances the solubility of protein and is more expressed in supernatant.
(2) The recombinant Cys C greatly promotes the protein solubility expression efficiency and enhances the protein stability.
(3) The codon sequence obtained by optimization can be expressed in a large amount in escherichia coli, and the expression quantity of the recombinant Cys C is obviously improved.
(4) The recombinant Cys C expressed in the escherichia coli is mainly soluble fusion protein, so that the purification process is simple.
(5) The recombinant Cys C of the invention expressed by prokaryotic recombination maintains excellent antigen performance.
The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1
1) The gene of human Cys C provided by NCBI is used as reference, the experimental design requirement of the invention is combined, the amino acid sequence shown in SEQ ID NO 1 is determined by a carrier without a label, after the preference of the Escherichia coli synonymous codon is optimized (SEQ ID NO:2), the connecting carrier is pET-28a (+) (C end fusion expression (His)6 label in the carrier), and the carrier is synthesized by Nanjing Kingsler Biotech Co., Ltd.
The amino acid sequence of the fusion Cys C protein with the label is shown as SEQ ID NO. 3, and the fusion Cys C protein cuts the first 26 amino acids at the N end of the SEQ ID NO. 1 so as to better promote the soluble expression of the Cys C protein. After optimization of Escherichia coli synonymous codon preference (SEQ ID NO:4), the ligation vector was pET-32a (+) (C-terminal fusion expression (His)6 tag in the vector) and synthesized by Nanjing Kingsler Biotech Co., Ltd.
2) Introduction of recombinant plasmid into host Escherichia coli
mu.L of the expression plasmid was taken and added to 30. mu.L of Escherichia coli competent Rosetta (DE3) under ice bath conditions, and the mixture was allowed to stand on ice bath for 20min, heat-shocked for 90s, immediately kept on ice for 2min, added to 400. mu.L of SOC medium without antibiotic, and cultured at 37 ℃ for 50min with shaking at 220 rpm.
100 μ L of the suspension was spread evenly on LB plates containing 100 μ g/mL kanamycin resistance and cultured overnight in a 37 ℃ incubator.
3) Process of expression of target gene
Picking the single clone in the step 2), inoculating the single clone in TB medium containing 100 mu g/mL kana resistance in a sterile operation, and culturing the single clone to OD at 37 ℃ by shaking at 220rpm600IPTG induction was performed between 0.6-0.8 and shaking culture was performed at 37 ℃ and 18 ℃ overnight, respectively.
Samples were sonicated for SDS-PAGE identification:
the predicted molecular weight of the Cys C protein without the label is 15.81KD, and the result in figure 1 shows that the Cys C protein is not soluble expressed in SB, TB and SOC culture media at 37 ℃ and 18 ℃;
the predicted molecular weight of the coupling label fused Cys C protein is 36.71KD, and as can be seen in figure 2, the coupling label Cys C protein can be soluble expressed in a large amount in the supernatant in the TB culture medium, and the expression amount accounts for more than 80% of the total protein of the thallus.
EXAMPLE 2 purification of Large amounts of expression products
Shake flask culture 1.5L bacterial liquid, centrifugal collection thalli wet weight: 26 g. About 4g of the cells were weighed and resuspended in 35ml of lysine Buffer on ice. Centrifuging after ultrasonic crushing at 20000rpm at 4 deg.C for 30min, collecting supernatant, and filtering with 0.22 μm needle filter to obtain filtered bacteria solution.
Filtering, performing Ni-column affinity chromatography, eluting with 50mM Tris-HCl, 50mM NaCl, 200mM imidazole pH7.0 to obtain target protein, eluting to obtain 7ml of protein with concentration of 2.13mg/ml, and electrophoretogram shown in FIG. 3.
The expression content of the target protein is calculated to be 97mg/L, and the purity can reach 95%.
Example 3 protein stability assay
The obtained target protein was dispensed into 2mL EP tubes, 1 mL/tube, and sealed with a sealing film.
3 of each batch, 1 of which was placed at 4 ℃ as a control and the remaining 4 at 37 ℃ for one week accelerated testing.
Samples were taken for identification at 3 and 7 days, respectively, and the stability of the protein was tested by UNcle test (Unchained Lab, usa, multifunctional protein stability analysis system).
The results in FIG. 4 show that the recombinant fusion Cys C protein has better stability after being placed at 37 ℃ for 3 and 7 days, and has no obvious difference from the recombinant fusion Cys C protein placed at 4 ℃.
Example 4 Elisa identification of immunological Activity
The purified fusion Cys C protein was added to 0.2. mu.g/well of the microplate and coated overnight at 4 ℃.
After coating, washing for 3 times by a plate washing machine, adding skimmed milk powder into a constant temperature incubator at 37 ℃, and sealing for 1 h.
Human Cys C protein polyclonal antibody (purchased from Darriy Biotechnology Ltd., Guangzhou) diluted with PBS at different 1K, 2K, 4K, 8K and 16K multiples was then added, incubated in a constant temperature incubator at 37 ℃ for 2h, and the plates were washed 3 times after completion.
Adding secondary antibody (purchased from Biotechnology engineering (Shanghai) Co., Ltd.) and incubating at 37 deg.C for 30min, washing for 5 times, tapping dry solution, adding TMB color developing solution, and developing in dark at room temperature for 10min until light blue can be seen.
Add stop solution 50 u L/hole, in the enzyme-linked immunosorbent assay 450nm/630nm OD value.
The results are shown in fig. 5, where the recombinantly expressed Cys C protein bound well to the antibody and was very immunogenic.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Guangzhou Daan Gene GmbH
<120> Process for producing recombinant cystatin C
<130> 000098
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 146
<212> PRT
<213> human (Homo sapiens)
<400> 1
Met Ala Gly Pro Leu Arg Ala Pro Leu Leu Leu Leu Ala Ile Leu Ala
1 5 10 15
Val Ala Leu Ala Val Ser Pro Ala Ala Gly Ser Ser Pro Gly Lys Pro
20 25 30
Pro Arg Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu Glu Glu Gly
35 40 45
Val Arg Arg Ala Leu Asp Phe Ala Val Gly Glu Tyr Asn Lys Ala Ser
50 55 60
Asn Asp Met Tyr His Ser Arg Ala Leu Gln Val Val Arg Ala Arg Lys
65 70 75 80
Gln Ile Val Ala Gly Val Asn Tyr Phe Leu Asp Val Glu Leu Gly Arg
85 90 95
Thr Thr Cys Thr Lys Thr Gln Pro Asn Leu Asp Asn Cys Cys Phe His
100 105 110
Asp Gln Pro His Leu Lys Arg Lys Ala Phe Cys Ser Phe Gln Ile Tyr
115 120 125
Ala Val Pro Trp Gln Gly Thr Met Thr Leu Ser Lys Ser Thr Cys Gln
130 135 140
Asp Ala
145
<210> 2
<211> 438
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
atggcaggac ccctaagggc tccattattg cttctggcga ttttggcggt agcgctggcg 60
gtgagcccgg cagcaggctc gagcccaggt aaaccgcctc gcttggtcgg tggtccgatg 120
gacgccagcg ttgaggaaga aggcgtgcgt cgtgcgctcg acttcgcagt tggtgaatac 180
aacaaagctt ctaatgatat gtatcatggc cgtgcgctgc aggttgttcg cgcacgtaag 240
caaatcgtgg ccggtgtgaa ttacttcctg gatgttgagt tgggccgtac gacctgtacc 300
aagacccaac cgaacctgga caactgcccg tttcacgatc agccgcacct gaaacgcaag 360
gcgttttgct ccttccaaat ctatgctgtg ccgtggcagg gcaccatgac tctgagcaag 420
tccacctgcc aggacgcg 438
<210> 3
<211> 240
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 3
Ser Ser Pro Gly Lys Pro Pro Arg Leu Val Gly Gly Pro Met Asp Ala
1 5 10 15
Ser Val Glu Glu Glu Gly Val Arg Arg Ala Leu Asp Phe Ala Val Gly
20 25 30
Glu Tyr Asn Lys Ala Ser Asn Asp Met Tyr His Ser Arg Ala Leu Gln
35 40 45
Val Val Arg Ala Arg Lys Gln Ile Val Ala Gly Val Asn Tyr Phe Leu
50 55 60
Asp Val Glu Leu Gly Arg Thr Thr Cys Thr Lys Thr Gln Pro Asn Leu
65 70 75 80
Asp Asn Cys Cys Phe His Asp Gln Pro His Leu Lys Arg Lys Ala Phe
85 90 95
Cys Ser Phe Gln Ile Tyr Ala Val Pro Trp Gln Gly Thr Met Thr Leu
100 105 110
Ser Lys Ser Thr Cys Gln Asp Ala Ser Ser Pro Gly Lys Pro Pro Arg
115 120 125
Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu Glu Glu Gly Val Arg
130 135 140
Arg Ala Leu Asp Phe Ala Val Gly Glu Tyr Asn Lys Ala Ser Asn Asp
145 150 155 160
Met Tyr His Ser Arg Ala Leu Gln Val Val Arg Ala Arg Lys Gln Ile
165 170 175
Val Ala Gly Val Asn Tyr Phe Leu Asp Val Glu Leu Gly Arg Thr Thr
180 185 190
Cys Thr Lys Thr Gln Pro Asn Leu Asp Asn Cys Cys Phe His Asp Gln
195 200 205
Pro His Leu Lys Arg Lys Ala Phe Cys Ser Phe Gln Ile Tyr Ala Val
210 215 220
Pro Trp Gln Gly Thr Met Thr Leu Ser Lys Ser Thr Cys Gln Asp Ala
225 230 235 240
<210> 4
<211> 720
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
tcaagtcccg gaaaaccacc taggctagta ggcggtccaa tggatgcctc tgttgaggaa 60
gagggcgtgc gtcgcgcgtt ggactttgca gttggtgaat ataacaaagc ttccaacgat 120
atgtaccata gccgtgcgct gcaagtaatg agagctcgca agcagattgt cgcgggtgta 180
aattacttct tggacgttga gctcggtcgt accacctgta ccaagaccca gccgaatctg 240
gacaactgcc cgtttcacga tcaaccgcac ctgaaacgat aggcgttctg ctcgttccaa 300
atctatgcag ttccgtggca gggcaccatg actctgagca aaagcacgtg ccaggacgcg 360
tcgagcccag gtaaaccgcc tcgcttggtc ggtggtccga tggacgccag cgttgaggaa 420
gaaggcgtgc gtcgtgcgct cgacttcgca gttggtgaat acaacaaagc ttctaatgat 480
atgtatcatg gccgtgcgct gcaggttgtt cgcgcacgta agcaaatcgt ggccggtgtg 540
aattacttcc tggatgttga gttgggccgt acgacctgta ccaagaccca accgaacctg 600
gacaactgcc cgtttcacga tcagccgcac ctgaaacgca aggcgttttg ctccttccaa 660
atctatgctg tgccgtggca gggcaccatg actctgagca agtccacctg ccaggacgcg 720

Claims (10)

1. A cystatin C fusion protein, wherein the cystatin C fusion protein is selected from the group consisting of:
(A) a polypeptide having an amino acid sequence shown in SEQ ID NO. 3;
(B) a polypeptide having a homology of not less than 90% (preferably, not less than 95% homology; etc., preferably, not less than 96% homology; most preferably, not less than 97% homology) with the amino acid sequence shown in SEQ ID NO. 3, and which retains the activity of the polypeptide shown in SEQ ID NO. 1;
(C) a derivative polypeptide which is formed by substituting, deleting or adding 1-5 amino acid residues of the amino acid sequence shown in SEQ ID NO. 3 and keeps the activity of the polypeptide shown in SEQ ID NO. 1.
2. The cystatin C fusion protein of claim 1, wherein the cystatin C fusion protein is isolated.
3. The cystatin C fusion protein of claim 1, wherein the amino acid sequence of the cystatin C fusion protein is shown in SEQ ID No. 3.
4. An isolated codon-optimized polynucleotide encoding the fusion protein of claim 1.
5. The polynucleotide of claim 4, wherein the polynucleotide is selected from the group consisting of:
(a) polynucleotide with sequence shown in SEQ ID NO. 4;
(b) polynucleotide having a nucleotide sequence homology of 95% or more (preferably 98% or more) with the sequence shown in SEQ ID NO. 4;
(c) a polynucleotide complementary to any one of the polynucleotides of (a) - (b).
6. An expression vector comprising the polynucleotide of claim 4.
7. A host cell comprising the expression vector of claim 6 or having the polynucleotide of claim 4 integrated into its genome.
8. The host cell of claim 7, wherein the host cell is a prokaryotic cell, preferably the host cell is E.coli, more preferably the E.coli Rosetta (DE3) strain.
9. A method of making the fusion protein of claim 1, comprising the steps of:
culturing the cell of claim 7 under conditions suitable for expression, thereby expressing the fusion protein of claim 1; and isolating the fusion protein.
10. A kit comprising the fusion protein of claim 1, the polynucleotide of claim 4, or the expression vector of claim 6, or the host cell of claim 7.
CN202111551776.9A 2021-12-17 2021-12-17 Method for preparing recombinant cystatin C Active CN114409800B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111551776.9A CN114409800B (en) 2021-12-17 2021-12-17 Method for preparing recombinant cystatin C

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111551776.9A CN114409800B (en) 2021-12-17 2021-12-17 Method for preparing recombinant cystatin C

Publications (2)

Publication Number Publication Date
CN114409800A true CN114409800A (en) 2022-04-29
CN114409800B CN114409800B (en) 2024-04-05

Family

ID=81267466

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111551776.9A Active CN114409800B (en) 2021-12-17 2021-12-17 Method for preparing recombinant cystatin C

Country Status (1)

Country Link
CN (1) CN114409800B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087622A (en) * 2015-07-08 2015-11-25 北京嘉万生物技术有限公司 Method for improving soluble expression quantity of human cystatin C protein in escherichia coli
CN109975552A (en) * 2017-12-28 2019-07-05 江苏众红生物工程创药研究院有限公司 A kind of recombination cystatin C albumen and its application in detection kit

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105087622A (en) * 2015-07-08 2015-11-25 北京嘉万生物技术有限公司 Method for improving soluble expression quantity of human cystatin C protein in escherichia coli
CN109975552A (en) * 2017-12-28 2019-07-05 江苏众红生物工程创药研究院有限公司 A kind of recombination cystatin C albumen and its application in detection kit

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"cystatin C [Homo sapiens]", GENBANK: AAA52164.1 *

Also Published As

Publication number Publication date
CN114409800B (en) 2024-04-05

Similar Documents

Publication Publication Date Title
CA2583009C (en) Ubiquitin or gamma-crystalline conjugates for use in therapy, diagnosis and chromatography
CN108660128B (en) Alfalfa terpene synthase, encoding gene, vector, polyclonal antibody and application thereof
CN111875676A (en) P49 mutant protein of African swine fever virus immunogen, recombinant vector, Escherichia coli genetic engineering bacteria, preparation method and application
CN111499759A (en) Zinc finger protein-lactoferrin fusion protein with cell membrane penetrating property and preparation and application thereof
KR102453605B1 (en) Novel Peptide Tag, Antibodies Binding to the Same and Uses thereof
CN110878123B (en) anti-TK 1 prokaryotic recombinant single-chain antibody and preparation method thereof
JP5865002B2 (en) Recombinant plasmid vector and protein production method using the same
CN114409800B (en) Method for preparing recombinant cystatin C
CN110938656A (en) Recombinant expression vector of giant panda follicle-stimulating hormone, expression system and preparation method
CN109320597B (en) Fox subfamily activin A protein and preparation and application thereof
CN114380920B (en) Human alpha fetoprotein fusion protein, and preparation method and application thereof
CN117720669A (en) Method for preparing recombinant cystatin C by eukaryotic cell expression
CN117720668A (en) Method for preparing human prealbumin
CN107434826B (en) Yeast cell for high expression of Slit2D2-HSA protein and application
CN109609481B (en) Preparation method of escherichia coli beta galactosidase receptor
CN109293763B (en) Mink activin B protein and preparation and application thereof
CN113480666B (en) CA153 fusion protein and preparation method thereof, and CA153 detection quality control product or calibrator
CN112689674B (en) Dextran affinity tag and application thereof
CN116396395A (en) Human transferrin fusion protein and preparation method thereof
CN113087807B (en) Shiga toxin B subunit recombinant protein-based probe for detecting carbohydrate antigen and preparation method thereof
CN116063571A (en) Preparation method and application of recombinant SSB antigen
CN115838437A (en) Human NT-proBNP fusion protein and preparation method and application thereof
CN110003334B (en) Polypeptide, CD19 single domain antibody, preparation method thereof, nucleotide sequence and kit
CN112979769B (en) Amino acid sequence, protein, preparation method and application thereof
CN114480346B (en) DNA hydrolase and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
CB03 Change of inventor or designer information
CB03 Change of inventor or designer information

Inventor after: Jiang Xiwen

Inventor after: Huang Hong

Inventor after: Huang Li

Inventor after: Yan Qingqing

Inventor after: Kuang Xiuli

Inventor after: Xiao Lanhua

Inventor before: Jiang Xiwen

Inventor before: Huang Hong

Inventor before: Yan Qingqing

Inventor before: Kuang Xiuli

Inventor before: Xiao Lanhua

GR01 Patent grant
GR01 Patent grant