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Method for expression and purification of recombinant CXCL9 (C-X-C motif chemokine 9) protein and application of method

A technology for expression, purification, and protein, which is applied in the field of soluble expression of recombinant CXCL9 protein by the comprehensive use of solubilizing tags and inteins, which can solve the problems of difficult large-scale production, low expression yield, etc. The effect of good application prospects

Active Publication Date: 2018-10-26
SHANGHAI JIAO TONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are still great difficulties in the preparation of recombinant CXCL9 protein. The expression yield in insect cells and mammalian cells is low, and it is difficult to achieve large-scale production. Although the expression level in the E. coli system is high, it is easy to form inclusion bodies. Biologically active products must be obtained through tedious denaturation and renaturation steps

Method used

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  • Method for expression and purification of recombinant CXCL9 (C-X-C motif chemokine 9) protein and application of method
  • Method for expression and purification of recombinant CXCL9 (C-X-C motif chemokine 9) protein and application of method
  • Method for expression and purification of recombinant CXCL9 (C-X-C motif chemokine 9) protein and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Construction of pET30a / Zbasic-ΔI-CM-CXCL9 plasmid and soluble expression of mCXCL9

[0051] In this example, we use Zbasic as a solubilizing tag to construct an Intein (ΔI-CM)-mediated fusion protein expression plasmid. Using PCR and restriction enzyme digestion and ligation methods, the sequences of Zbasic, ΔI-CM, and mCXCL9 are sequentially connected and inserted simultaneously In the pET30a plasmid, a flexible peptide chain is used between the tag coding sequence and the intein coding sequence, and a separate PET30a / mCXCL9 expression plasmid is constructed as a control. The construction method is as follows figure 2 Shown.

[0052] After the plasmid was successfully constructed, the BL21(DE3) host bacteria were transformed, and the monoclonal colonies were picked and inoculated into Kana+'s LB liquid culture, and cultivated at 37°C and 200rpm for 12 hours as seed bacteria. The seed bacteria were inoculated in sterile Kana+LB medium at a volume ratio of 1:100, cultured at...

Embodiment 2

[0056] Use other solubilizing labels to achieve soluble expression of mCXCL9

[0057] In this example, we replaced Zbasic with two other dissolution-promoting tags to prove the extensibility of the method of the present invention. We use two tags, FATT (SEQ ID NO.5) and Fh8 (SEQ ID NO.6), and use PCR and restriction enzyme ligation methods to connect the solubilizing tags, ΔI-CM, and mCXCL9 sequences and insert them into the pET30a vector. , Obtain the pET30a / FATT-ΔI-CM-CXCL9 and pET30a / Fh8-ΔI-CM-CXCL9 plasmids, and transform the BL21(DE3) host bacteria, perform induction expression and SDS-PAGE and Western blot detection according to the steps in Example 1, Such as Figure 4 Shown.

[0058] The detection conditions were as follows: 37℃ induced FATT-ΔI-CM-CXCL9 and Fh8-ΔI-CM-CXCL9 expression, separated soluble supernatant and inclusion body components for SDS-PAGE (A) and Western blot (B). Lane M: Marker; Lane Ind: Induced bacterial solution; Lane Sup: Supernatant after fission; ...

Embodiment 3

[0061] Purification of mCXCL9 after soluble expression

[0062] After the soluble expression of mCXCL9 protein is obtained by the method of the present invention, it is suitable for purification by cation exchange chromatography due to its high isoelectric point (PI=10.62). The specific method is that the host bacteria expressing m recombinant CXCL9 protein are resuspended to a concentration of 10% with a solution of 50 mM PB, pH 7.0, and then crushed by high pressure homogenization, and centrifuged to obtain a supernatant containing soluble mCXCL9. Select SP FF medium for cation exchange purification, first use SPFF Loading Buffer (50mM PB, 1mM EDTA, 100 μM PMSF, pH 7.0), equilibrate the column at a flow rate of 1mL / min, and continue 1 mL / min loading after the UV baseline is stable , Make the target protein bind to SP FF medium. After loading the sample, continue to use LoadingBuffer to clean the column and adjust the solvent to pH 9.0 for elution. Finally, the target protein ...

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Abstract

The invention relates to the technical field of biology, in particular to a method for achieving soluble expression of recombinant CXCL9 (C-X-C motif chemokine 9) protein in Escherichia coli by fusionof a solubilizing label and Intern. The target protein is expressed by fusion with the solubilizing label and the Intein with the self-shear function, and soluble protein and shear release of the target protein are realized. Compared with a known method of expression of recombinant CXCL9 protein in Escherichia coli, the method has the advantages that the process of inclusion volume degeneration renaturation can be avoided, introduction of protease is not needed, a target product with the completely naturally sequence can be obtained, and good application prospect for the enlarged-scale industrial production is achieved.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a method and application for comprehensively using solubilizing tags and intein to realize soluble expression of recombinant CXCL9 protein. Background technique [0002] Recombinant protein products such as enzymes, proteins and antibodies play an indispensable role in people’s production, life, and medical treatment. In particular, biotechnology drugs such as recombinant proteins and monoclonal antibodies were developed in the late 20th century with the development of life sciences. The latest generation of drugs that have emerged have a series of advantages such as high efficiency, low toxicity, and clear biological functions. [0003] However, biotechnology drugs represented by recombinant proteins and polyclonal antibodies have large molecular weights and complex structures, and there are still considerable technical difficulties in expression, purification, and quality control....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/52C07K1/22C07K1/18C12N15/70
CPCC07K14/521C12N15/70
Inventor 路慧丽朱建伟罗晗张浩王燕石伟陆吉麟
Owner SHANGHAI JIAO TONG UNIV
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