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A method for expressing and purifying recombinant cxcl9 protein and its application

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. cost reduction effect

Active Publication Date: 2021-11-02
SHANGHAI JIAO TONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A method for expressing and purifying recombinant cxcl9 protein and its application
  • A method for expressing and purifying recombinant cxcl9 protein and its application
  • A method for expressing and purifying recombinant cxcl9 protein and its application

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, and use PCR and enzyme-cut ligation methods to simultaneously ligate and insert the sequences of Zbasic, ΔI-CM, and mCXCL9 in sequence. In the pET30a plasmid, a flexible peptide chain is used between the tag coding sequence and the intein coding sequence, and a separate expression plasmid of PET30a / mCXCL9 is constructed as a control. The construction method is as follows: figure 2 shown.

[0052] After the successful construction of the plasmid, transform the BL21(DE3) host bacterium, pick a single clone colony and inoculate it in the LB liquid culture of Kana+, and cultivate it at 200 rpm at 37°C for 12 hours as the seed bacterium. The seed bacteria were inoculated in sterile Kana+LB medium at a volume ratio of 1:100, cultured at 37°C and 200 rpm for ...

Embodiment 2

[0056] Soluble expression of mCXCL9 achieved with additional lytic tags

[0057] In this example, we replaced Zbasic with two other solubilizing tags to verify the extension of the method of the present invention. We selected two tags, FATT (SEQ ID NO.5) and Fh8 (SEQ ID NO.6), and used PCR and enzyme-cut ligation methods to sequentially connect the sequences of the lytic tag, ΔI-CM, and mCXCL9 and insert them into the pET30a vector , obtain pET30a / FATT-ΔI-CM-CXCL9 and pET30a / Fh8-ΔI-CM-CXCL9 plasmids, and transform BL21(DE3) host bacteria, induce expression and detect by SDS-PAGE and Western blot as in the steps of Example 1, Such as Figure 4 shown.

[0058] The detection conditions were as follows: FATT-ΔI-CM-CXCL9 and Fh8-ΔI-CM-CXCL9 were induced to express at 37°C, and the soluble supernatant and inclusion body components were separated for SDS-PAGE (A) and Western blot (B). Lane M: Marker; Lane Ind: Bacteria solution after induction; Lane Sup: Supernatant after lysis; L...

Embodiment 3

[0061] mCXCL9 purification after soluble expression

[0062] After the soluble expression of the mCXCL9 protein is obtained by the method of the present invention, it is suitable to be purified by cation exchange chromatography because of its high isoelectric point (PI=10.62). The specific method is as follows: host bacteria expressing m recombinant CXCL9 protein are resuspended to 10% concentration with 50mM PB, pH 7.0 solution, homogeneously broken by high pressure, and centrifuged to obtain supernatant containing soluble mCXCL9. Select SPFF medium for cation exchange purification. First, use SPFF Loading Buffer (50mM PB, 1mM EDTA, 100μM PMSF, pH 7.0) to equilibrate the chromatography column at a flow rate of 1mL / min. After the UV baseline is stable, continue to load the sample at 1mL / min. The protein of interest is bound to the SP FF medium. After loading the sample, continue to wash the column with Loading Buffer, and adjust the solvent to pH 9.0 for elution. Finally, th...

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Abstract

The invention relates to the field of biotechnology, and specifically discloses a method for realizing soluble expression of recombinant CXCL9 protein in Escherichia coli by using fusion expression of a solubilization tag and an intein (Intein) and an application thereof. In the method, the target protein is fused and expressed with a solubilizing tag and an Intein with self-cleavage function, so as to realize the soluble expression and shear release of the target protein. Compared with the known method for expressing recombinant CXCL9 protein in Escherichia coli, the method of the present invention can avoid the denaturation and renaturation process of inclusion bodies, does not need to introduce protease, and can obtain a target product with a completely natural sequence, which is useful for large-scale industrial production Very good application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for realizing the soluble expression of recombinant CXCL9 protein by comprehensively utilizing a solubilization tag and an intein and an application thereof. 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 with the development of life sciences in the late 20th century. The latest generation of drugs that have emerged has 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, purifica...

Claims

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Application Information

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Patent Type & Authority Patents(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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