Protein simple purification method for positioning recombinant protein to surfaces of cells

A recombinant protein, cell surface technology, applied in the field of simple purification of protein, to achieve the effect of simple operation, saving time and cost

Active Publication Date: 2020-07-24
上海百英生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the defect in the prior art that recombinant proteins require complex purification steps to obtain relatively pure proteins after they are expressed in cells, the technical problem to be solved in this application is to provide a method for quickly obtaining high-purity proteins without lysing cells and purifying through chromatographic columns. The method of the target protein, the method is to fuse and express the gene encoding the cell membrane anchor protein (signal peptide), the chemical method cleavage site and the target protein, so that the fusion protein is positioned on the surface of the cell

Method used

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  • Protein simple purification method for positioning recombinant protein to surfaces of cells
  • Protein simple purification method for positioning recombinant protein to surfaces of cells
  • Protein simple purification method for positioning recombinant protein to surfaces of cells

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1: Construction and expression of Lpp-OmpA-(SNAC-tag)-GB1 fusion protein and Lpp-OmpA-(SNAC-tag)-SFGFP fusion protein

[0045] The mode of the fusion protein used in this embodiment is as follows figure 1 As shown, the code Lpp(1-9)-OmpA(45-159)-(SNAC-tag)-GB1 (sequence shown in SEQ ID NO.1) obtained by gene synthesis (Sangon Bioengineering Co., Ltd.) Shown) the pet30a plasmid was transformed into E.coli BL21(DE3), the expression was induced by 0.5mM IPTG at 30°C for 5h, and the bacteria were harvested by a high-speed centrifuge. and stored at -80°C until use. The pet30a plasmid encoding Lpp(1-9)-OmpA(45-159)-(SNAC-tag)-SFGFP (sequence shown in SEQ ID NO.2) was obtained by total gene synthesis (Sangon Bioengineering Co., Ltd.) Express in the same way and collect bacteria.

Embodiment 2

[0046] Embodiment 2: Construction, expression and purification of 6His-GB1 fusion protein and 6His-SFGFP fusion protein

[0047] The pet28a plasmid encoding 6His-GB1 (sequence shown in SEQ ID NO.3) obtained by gene synthesis (Sangon Bioengineering Co., Ltd.) was transformed into E.coli BL21 (DE3), using 0.5mM IPTG in The expression was induced at 30°C for 5 hours, and the bacteria were harvested using a high-speed centrifuge. and stored at -80°C until use. The pet28a plasmid encoding 6His-SFGFP (sequence shown in SEQ ID NO.4) was obtained by total gene synthesis (Sangon Bioengineering Co., Ltd.) and obtained and expressed in the same manner, and the bacteria were harvested.

Embodiment 3

[0048] Example 3: Identification of cell membrane and cytoplasmic components of bacteria expressing fusion protein

[0049] In order to characterize whether the signal peptide can localize the target protein to the cell membrane, it is necessary to identify the cell membrane and cytoplasmic components of the bacteria expressing the recombinant protein by SDS-PAGE gel. Bacteria were resuspended in 10 mM PBS (pH 7.4) and lysed using sonication. The supernatant and the precipitate were separated by high-speed centrifugation at 12000 rpm, and the precipitate was eluted twice with TDSET buffer (1% Triton X-100, 0.2% sodium deoxycholate, 0.1% SDS, 10 mM tetrasodium EDTA, and 10 mM Tris / HCl). Pellets and supernatants were identified using SDS-PAGE. figure 2 The 2nd lane is the supernatant of bacteria expressing Lpp-OmpA-(SNAC-tag)-GB1, the 3rd lane is the cell membrane components of Lpp-OmpA-(SNAC-tag)-GB1 bacteria, and the 4th lane is expressing 6His- The supernatant of GB1 bacte...

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Abstract

The invention discloses a protein simple purification method for positioning recombinant protein to the surfaces of cells. The method is characterized by comprising the following steps: constructing an encoding signal peptide, a chemical cleavage site and a target protein gene into an expression vector, and positioning target protein to the surfaces of the cells after being expressed; and adding abuffer solution containing a cleavage reagent into the collected cells for resuspension, and performing centrifugation to obtain the supernatant, so as to obtain a target protein solution. Accordingto the protein simple purification method disclosed by the invention, the recombinant protein is secreted and positioned to the surface of a cell after being expressed in the cell by utilizing the positioning function of signal peptide, and by utilizing the advantages of intermediate chemical shearing sites of the recombinant protein, the high-purity target protein can be simply and conveniently obtained by adding a cleavage reagent into a buffer solution for resuspending cells and centrifuging. The method omits complex steps that in the prior art, protein purification needs cell lysis and chromatographic columns with various properties are used to obtain target protein, is simple to operate, and greatly saves the time and costs.

Description

technical field [0001] The application belongs to the field of protein separation and purification, and relates to a simple method for purifying protein by positioning recombinant protein on the cell surface. By constructing the fusion gene encoding the signal peptide, the chemical cleavage site and the target protein into the expression vector, the target protein can be localized to the outer membrane of the cell after expression. The recombinant protein expressed by this method only needs to be resuspended in the buffer containing the cleavage reagent in the collected cells, without lysing the cells and purifying the chromatographic column, and can obtain the target protein with high purity and high yield in one step after centrifugation. Background technique [0002] The expression systems of recombinant proteins can be divided into four types: prokaryotic, mammalian, yeast and insect expression systems. The expression sites of recombinant proteins in cells can be divide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C12P21/00C07K1/14
CPCC07K14/00C07K2319/02C07K2319/21C12N15/70C12P21/00
Inventor 黄善青沙万星刘天意魏天彪
Owner 上海百英生物科技股份有限公司
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