CN117327732A - Expression plasmid combination, expression host cell and expression method for fully self-cleaving recombinant C1s protein - Google Patents
Expression plasmid combination, expression host cell and expression method for fully self-cleaving recombinant C1s protein Download PDFInfo
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- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21041—Complement subcomponent C1r (3.4.21.41)
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- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21042—Complement subcomponent C1s (3.4.21.42)
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Abstract
The invention discloses an expression plasmid combination for fully self-cleaving recombinant C1s protein. The combination contains two plasmids which respectively contain the expression gene of the C1s protein and the expression gene of the C1r protein, wherein the C terminal of the C1s protein encoded by the expression gene of the C1s protein carries a purification tag, and the C1r protein encoded by the expression gene of the C1r protein does not introduce the purification tag. The invention also discloses an expression host cell and an expression method thereof. The invention uses the cascade mechanism of C1s protein, and co-transfers and expresses the upstream C1r protein while expressing the C1s protein, thereby introducing the mechanism of specific activation C1s protein into host cells to obtain the fully self-cracked recombinant C1s protein. Using this method, fully cleaved C1s proteins can be obtained by preparation using a laboratory common mammalian cell expression system (e.g., HEK 293).
Description
Technical Field
The invention relates to an expression plasmid combination, an expression host cell and an expression method for fully self-cleaving recombinant C1s protein, and belongs to the technical field of protein expression.
Background
The recombinant protein expression technology is to insert exogenous gene encoding target protein into expression vector through molecular cloning, then to infect host cell with the expression vector, to produce protein through the transcription and translation mechanism of the host cell, and to obtain target protein through separation and purification. Common host cells for expression include E.coli, yeast, insect and mammalian cells, and the like.
The C1s protein consists of two subunits, of which the B subunit is a serine protease. The C1s protein is one of the important components of the system of the protein cascade in the complement system, and forms the C1 complement system together with C1q and C1r, which are important components of the immune mechanism of the body. The C1r protein activates the C1s protein, releases the protease activity, and then the C1s protein further activates the C2 and C4 proteins in the complement system, so as to complete the cascade reaction of the complement system. The C1s protein is one of important drug targets, and the deletion thereof can lead to various immune related diseases, including Ehlers-Danlos syndrome, hashimoto thyroiditis, hepatitis and the like. Therefore, the C1s protein has important research value. The C1s proteins for scientific research are usually prepared by recombinant protein expression techniques, and the main host used is mammalian cells. The common mammalian cell expression system (such as HEK 293) host cells contain a certain background protease, so that the C1s protein can be partially cleaved and activated, however, the expressed C1s protein cannot be completely cleaved and activated usually due to poor specificity of the background protease of the host cells, and the proportion of batch-to-batch protein activation is large in difference, so that the completely activated C1s protein with biological significance cannot be prepared. The current preparation of fully activated C1s proteins of sufficient biological significance requires the use of special cell lines, such as the mouse myeloma cell line NS0, which presents a significant challenge for the general laboratory to obtain the C1s proteins for research.
Disclosure of Invention
The invention aims to provide an expression plasmid combination for fully self-cleaving recombinant C1s protein, which can be directly cultured to obtain activated C1s protein.
The invention adopts the technical scheme that:
an expression plasmid combination for fully self-cleaving recombinant C1s protein, wherein the plasmid I contains an expression gene of the C1s protein, the plasmid II contains an expression gene of the C1r protein, wherein the C terminal of the C1s protein encoded by the expression gene of the C1s protein carries a purification tag, and the C1r protein encoded by the expression gene of the C1r protein does not introduce the purification tag.
Preferably, the purification tag is a His tag.
Preferably, the amino acid sequence of the C1s protein is shown in SEQ ID No.2, and the amino acid sequence of the C1r protein is shown in SEQ ID No. 4.
The invention also discloses an expression host cell for fully self-cleaving recombinant C1s protein, and a mammalian cell line transfected with the two expression plasmids.
Preferably, the mammalian cell line is HEK293 cells.
The invention also discloses an expression method of the fully self-cleaving recombinant C1s protein, which comprises the following steps:
(1) Constructing recombinant protein expression plasmids: constructing the expression plasmid combination of any one of claims 1-3;
(2) Transfecting the constructed two recombinant protein expression plasmids into a mammalian cell line, and culturing the transfected mammalian cell line;
(3) Collecting the supernatant of the culture solution, passing through a nickel ion affinity chromatography column, eluting, and collecting the eluting peak containing the target protein to obtain the C1s protein.
Preferably, the mammalian cell line is HEK293 cells.
Preferably, the eluent is 20mMTris,pH7.4, 150mMNaCl,1M imidazole.
Preferably, the step (2) specifically comprises: subculturing HEK293 cells with serum-free CD medium, and regulating cell density to 0.5-1X 10 6 When the recombinant protein expression plasmid is constructed, the constructed recombinant protein expression plasmid and a transfection reagent are mixed according to the mass ratio of 1:1, then adding the mixture into cells for transfection, and adding 293 serum-free feed liquid for cell culture on days 1,3 and 5 after transfection.
Preferably, after at least 7 days of incubation, the culture supernatant is collected, filtered and applied to a nickel ion affinity chromatography column, followed by equilibration with 10mM imidazole-containing Tris buffer, elution with a linear imidazole concentration gradient after equilibration is complete, and the elution peak containing the target protein is collected; tris buffer containing 10mM imidazole is 20mMTris,pH7.4, 150mMNaCl,10mM imidazole; the elution buffer was 20mMTris,pH7.4, 150mMNaCl,1M imidazole.
The invention uses the cascade mechanism of C1s protein, and co-transfers and expresses the upstream C1r protein while expressing the C1s protein, thereby introducing the mechanism of specific activation C1s protein into host cells to obtain the fully self-cracked recombinant C1s protein. Using this method, fully cleaved C1s proteins can be obtained by preparation using a laboratory common mammalian cell expression system (e.g., HEK 293).
Drawings
Fig. 1: c1s protease activity calculation formula.
Fig. 2: original process, single turn C1s expression plasmid, protein cleavage activation after purification. Left: SDS-PAGE and WB examined activation of recombinant C1s proteins. The C1s protein breaks into two AB subunits after activation, and is linked by interchain disulfide bonds, so that incompletely cleaved C1s proteins appear as one band on non-reducing PAGE and three bands on reducing PAGE. Right: relationship between the degree of activation of the C1s protein and serine hydrolase activity. In the figure, r=reduced SDS-PAGE electrophoresis, nr=non-reduced SDS-PAGE electrophoresis, and WB is a western blot reduced SDS-PAGE gel electrophoresis picture using an anti-C1 s antibody.
Fig. 3: cotransformation of C1s expression plasmid and C1r helper plasmid, and activation of protein cleavage after purification. Left: SDS-PAGE detects activation of recombinant C1s proteins. Right: fully activated C1s protein serine hydrolase activity. In the figure, r=reduced SDS-PAGE electrophoresis, nr=non-reduced SDS-PAGE electrophoresis.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way.
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, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention.
The materials or instruments used in the following examples, if not specifically described, were available from conventional commercial sources.
Example 1
Construction of recombinant protein expression plasmid: the recombinant DNA technology is used to integrate the genes of C1s and C1r proteins into the expression vector of mammal cell, and the sequence correctness of the inserted genes is verified by sequencing method. cDNA sequences encoding the C1s and C1r proteins were prepared by total gene synthesis and constructed onto pCDNA3.4 expression vectors (map reference https:// www.snapgene.com/resources/plastics-files/. The C-terminal end of the C1s protein carries a purification tag, and the purification tag is not introduced in the construction of the C1r protein, so that the C1s protein is purified by using an affinity chromatography method at a later stage. The cDNA sequence of the coded C1s protein is shown as SEQ1, and the amino acid sequence obtained after translation is shown as SEQ2; the cDNA sequence of the coded C1r protein is shown as SEQ3, and the amino acid sequence obtained after translation is shown as SEQ4.
Recombinant protein expression: subculturing HEK293 cells with serum-free CD medium (product No. SMM293-TI, yinqiao Shenzhou) to adjust the cell density to 0.5-1X 106, and mixing the C1s protein expression plasmid and the C1r protein expression plasmid according to the mass ratio of 1:1, and then mixed with a transfection reagent TF1 (Yiqiao Shenzhou, cat. Number STF 02) and subsequently added to the cells for transfection. 293 serum-free stock solution (Yiqiao Shenzhou, cat. No. M293-SUPI-100) was added 1,3,5 days after transfection. Protein purification was performed after 7 days of cell culture.
Purification of recombinant C1s protein: after 7 days of incubation, the culture supernatant was collected, filtered and applied to a nickel ion affinity column followed by equilibration using 10mM imidazole in Tris buffer (20mMTris,pH7.4, 150mMNaCl,10mM imidazole). After equilibration, the elution peak containing the target protein was collected using a linear imidazole concentration gradient. The elution buffer was 20mM Tris,pH7.4, 150mM NaCl,1M imidazole. Purity and cleavage activation extent of purified C1s protein were characterized using reduced and non-reduced SDS-PAGE. The results are shown in FIG. 3.
Recombinant C1s protease activity assay:
reagents and materials:
test buffer = 50mM Tris,250mM NaCl,pH8.0
Test substrate = N-benzyloxy lysine thiobenzyl ester (Z-K-SBzl, bachem cat# M-1300), dissolved in DMSO to make 10mM stock solution
DTNB = 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB, sigma cat No. D-8130), dissolved in DMSO to prepare a 10mM stock solution
Material = 96 well plate (Costar cat No. 92592), reader (SpectraMax Plus, molecular Devices)
The testing method comprises the following steps:
1. recombinant C1s was diluted to 0.2 ng/. Mu.L with test buffer
2. The test buffer solution for test substrate and the DTNB mother liquor were prepared into a working solution containing 200. Mu.M of test substrate and 200. Mu.M of DTNB
3. To the sample wells of the 96-well plate, 50. Mu.L of 0.2 ng/. Mu.L of recombinant C1s protein was added, and 50. Mu.L of 200. Mu.M substrate working solution was added, and the substrate working solution addition time point was the reaction start time. As a control, 50 μl of 200 μl of substrate working solution and 50 μl of test buffer were added to another sample of the well plate.
4. The reaction was carried out for 5 minutes after the addition of the substrate, and the change in fluorescence signal was continuously read at an absorbance value of 405nm using the kinetic mode of the plate reader.
5. Enzyme specific activity calculation: the formula is shown in fig. 1, and the results are shown in fig. 2 and 3.
Comparative example 1
The recombinant protein expressed as C1s protein was transfected into HEK293 cells alone and the C1s protein was expressed and purified by the same procedure as in example 1, the cDNA sequence encoding the C1s protein was shown in SEQ ID No.1, and the amino acid sequence obtained after translation was shown in SEQ ID No.2. The results are shown in FIG. 2.
It can be seen that C1s alone expressed in HEK293 cells, the protein had some cleavage by the host protease, however complete cleavage activation of the protein was not achieved (fig. 2, left). The protease activity of C1s increased with increasing proportion of proteolytic activation (fig. 2, right), and the same process was used for three separate runs of C1s protein expression in batches 1-3, and a significant difference in C1s protein expression between the three runs was seen, which resulted from incomplete post-treatment of the expressed C1s protein by the host cell. Therefore, the post-treatment of the C1s protein by the original process is incomplete and has poor reproducibility.
The production method using co-transformation of C1s and C1r resulted in a complete cleavage activated C1s protein (FIG. 3, left side) with a significantly increased protease activity compared to the partially cleavage activated C1s protein produced using the original process (FIG. 3, right side). The method is high in reproducibility, the C1s protein which is completely activated by cleavage and has equivalent enzyme catalytic activity is obtained by repeatedly producing the same method twice, the difference of the two enzyme activities is small, and the process is stable (figure 3, right side). The original process in fig. 3 is the process of comparative example 1.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. An expression plasmid combination for fully self-cleaving recombinant C1s protein is characterized in that a plasmid I contains an expression gene of the C1s protein, a plasmid II contains an expression gene of the C1r protein, wherein the C terminal of the C1s protein encoded by the expression gene of the C1s protein carries a purification tag, and the C1r protein encoded by the expression gene of the C1r protein is not introduced into the purification tag.
2. The expression plasmid combination according to claim 1, characterized in that the purification tag is a His tag.
3. The expression plasmid combination according to claim 1, wherein the amino acid sequence of the C1s protein is shown in SEQ ID No.2 and the amino acid sequence of the C1r protein is shown in SEQ ID No. 4.
4. An expression host cell that substantially self-cleaves a recombinant C1s protein, characterized by: a mammalian cell line transfected with two expression plasmids according to any of claims 1-3.
5. The expression host cell of claim 4, wherein the mammalian cell line is a HEK293 cell.
6. A method for expressing a substantially self-cleaving recombinant C1s protein, comprising the steps of:
(1) Constructing recombinant protein expression plasmids: constructing the expression plasmid combination of any one of claims 1-3;
(2) Transfecting the constructed two recombinant protein expression plasmids into a mammalian cell line, and culturing the transfected mammalian cell line;
(3) Collecting the supernatant of the culture solution, passing through a nickel ion affinity chromatography column, eluting, and collecting the eluting peak containing the target protein to obtain the C1s protein.
7. The method for expressing a substantially self-cleaving recombinant C1s protein according to claim 6, wherein: the mammalian cell line is HEK293 cells.
8. The method for expressing a substantially self-cleaving recombinant C1s protein according to claim 6, wherein: the eluent was 20mMTris,pH7.4, 150mMNaCl,1M imidazole.
9. The method for expressing a substantially self-cleaving recombinant C1s protein according to claim 6, wherein: the step (2) comprises the following steps: subculturing HEK293 cells by using a serum-free CD culture medium, and regulating the cell density to 0.5-1X 106, wherein the mass ratio of the constructed recombinant protein expression plasmid to the transfection reagent is (1): 1, then adding the mixture into cells for transfection, and adding 293 serum-free feed liquid for cell culture on days 1,3 and 5 after transfection.
10. The method for expressing a substantially self-cleaving recombinant C1s protein according to claim 6, wherein: the step (3) is specifically as follows: after at least 7 days of culture, collecting culture supernatant, filtering, loading the culture supernatant to a nickel ion affinity chromatography column, then balancing the culture supernatant by using a Tris buffer containing 10mM imidazole, eluting the culture supernatant by using a linear imidazole concentration gradient after balancing, and collecting elution peaks containing target proteins; tris buffer containing 10mM imidazole is 20mMTris,pH7.4, 150mMNaCl,10mM imidazole; the elution buffer was 20mMTris,pH7.4, 150mMNaCl,1M imidazole.
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| Application Number | Priority Date | Filing Date | Title |
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| US20100055106A1 (en) * | 2006-06-15 | 2010-03-04 | Eastern Virginia Medical School | Methods for regulating complement cascade proteins using astrovirus coat protein and derivatives thereof |
| CN112313249A (en) * | 2018-04-13 | 2021-02-02 | 中外制药株式会社 | Anti-complement component antibodies and methods of use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20100055106A1 (en) * | 2006-06-15 | 2010-03-04 | Eastern Virginia Medical School | Methods for regulating complement cascade proteins using astrovirus coat protein and derivatives thereof |
| CN112313249A (en) * | 2018-04-13 | 2021-02-02 | 中外制药株式会社 | Anti-complement component antibodies and methods of use |
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