Preparation of recombinant polypeptide by intein with trans-splicing function

A protein intron and trans-splicing technology, which is applied in the field of recombinant polypeptide preparation, can solve the problems such as the inability of protein introns to ligand and reduce the proportion of target polypeptide, and achieve the effects of simple structure, increased proportion and increased yield.

Inactive Publication Date: 2010-11-17
NANJING UNIV
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  • Summary
  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

(3) The intein itself cannot be used as a ligand for affinity purification
In order to improve the purification efficiency of the fusion protein, an affinity purification tag must be attached to the N-terminal or C-terminal of the intein, which further reduces the proportion of the target polypeptide in the fusion protein

Method used

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  • Preparation of recombinant polypeptide by intein with trans-splicing function
  • Preparation of recombinant polypeptide by intein with trans-splicing function
  • Preparation of recombinant polypeptide by intein with trans-splicing function

Examples

Experimental program
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Embodiment Construction

[0057] Preparation of recombinant Erythrina trypsin inhibitor (ETI):

[0058] 1. Construction of pET28a / His-inteinN and pET28a / inteinC-ETI expression plasmids

[0059] Construction of pET28a / His-inteinN expression plasmid: Using pTwin1 plasmid as a template, the gene sequence (inteinN) encoding the N-terminal splicing domain of the Ssp DnaB intein was amplified by PCR. PCR amplification conditions were: 5.0 fmol of pTwinl plasmid, 50 pmol of upstream and downstream primers, 0.25 U pyrobest enzyme, denaturation at 94°C for 2 minutes, followed by 26 cycles: 94°C, 30s; 55°C, 1min; 72°C, 60s, After purified by agarose electrophoresis, the PCR product was recovered with a DNA fragment recovery kit, digested with NdeI / XhoI double enzymes, recovered a large fragment (about 550bp), mixed with the expression plasmid pET28a digested by NdeI / XhoI, added T4 DNA ligase, and used The ligation product was transformed into E.coli DH5α competent cells, positive clones were identified by NdeI / ...

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Abstract

The invention provides preparation of recombinant polypeptide by an intein with a trans-splicing function, wherein the N-end splicing structure domain and the C-end splicing structure domain of the intein with the trans-splicing function can be specifically bound together. One splicing structure domain (the N- or C-end splicing structure domain) of the intein is taken as carrier protein to carry out fusion expression with target polypeptide; the other splicing structure domain (the C- or N-end splicing structure domain) is cross-linked with a supporting medium to prepare an affinity column for adsorbing and purifying the obtained fusion protein containing the target polypeptide; impurities in a fusion protein sample are removed by increasing the salinity of washing liquid in the affinity chromatographic column; and trans-splicing of the intein is induced by changing the pH and the temperature of the chromatographic column or adding a chemical reagent containing a sulfydryl group so as to release the target polypeptide from the fusion protein and purify the target polypeptide at the same time.

Description

1. Technical field [0001] The invention belongs to the field of recombinant polypeptide preparation. Utilize the specific interaction between the N-terminal splicing domain and the C-terminal splicing domain of the intein with trans-splicing activity to affinity purify the fusion protein containing the target polypeptide by changing the pH of the washing solution, Salt concentration, temperature, or the addition of chemical reagents containing sulfhydryl groups induce trans-cleavage of introns to release the target polypeptide from the fusion protein for purification. 2. Background technology [0002] With the in-depth study of genomics and proteomics, more and more proteins have been discovered. In order to study the biological functions of these proteins, it is first necessary to obtain a sufficient amount of high-activity, high-purity samples. Because the protein components in biological tissues are very complex, and the content of target proteins is often very small in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/30B01D15/08C07K1/22
Inventor 刘建宁孙自勇陆嵬
Owner NANJING UNIV
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