Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Prokaryotic expression vector and application thereof

A prokaryotic expression and carrier technology, applied in the field of bioengineering, can solve the problems of loss of target protein, failure to effectively increase the yield of target protein, effective folding of soluble expression, etc.

Inactive Publication Date: 2011-02-09
GUANGDONG MEDICAL UNIV
View PDF1 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage of fusion expression is that fusion tags and molecular chaperones are often removed by specific proteases, such as enterokinase, fXa, etc., after digestion or chemical cleavage.
This method often results in a large loss of pre-cleaved target protein in the process of fusion protein expression and its separation and purification.
At the same time, inteins cannot effectively increase the yield, soluble expression and promote the effective folding of the target protein.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Prokaryotic expression vector and application thereof
  • Prokaryotic expression vector and application thereof
  • Prokaryotic expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] The construction of embodiment 1 expression vector pICET32

[0019] The starting vector is pET32a, which has a high-efficiency T7 promoter and contains thioredoxin TrxA. It is one of the commonly used vectors to promote high-efficiency protein soluble expression. Design upstream primer DP1: 5'-CA TGGCCA TATGAAAATCGAAGAAGGTAAAC-3' (the underlined part in italics is the restriction endonuclease Msc I site) and downstream primer DP2: 5'-GTCCATGGCCGTTGTGTACAATGATGTCATTC-3' were amplified from the expression vector pTWIN1 (product of NEB Company) with pfu enzyme to obtain several The nucleotide sequence encoding the butyrin-binding domain (CBD) and the intein mini SspDnaB was used as a template to amplify the second-round product with the upstream primer DP1 and the newly designed primer 5'-GTACACAACGGCCATGGACATCATCATCATCATCATGGATCC-3'. A restriction endonuclease NcoI cutting site and a 6×his tag coding sequence were introduced through this round of amplification. Using t...

Embodiment 2

[0021] Example 2 Expression of hookworm anticoagulant peptide AcaNAP10 by pICET32 and its isolation and purification

[0022] According to the coding sequence of hookworm anticoagulant peptide AcaNAP10 (SEQ ID NO.1), primers were designed to amplify the coding nucleic acid sequence from the adult cDNA of hookworm, and the primer sequence was designed as: N10-3: 5'-GAGGATCCAATCCAAGCTGTGGTGAG-3' (including Dicer site BamH I); N11-2: 5'-CGAAGCTTGGTCATTTTCTATTAGGG-3' (contains endonuclease site Hind III). After the PCR product was recovered and purified, it was digested with BamH I and Hind III, and then ligated overnight with the expression plasmid pICET32 that had undergone the same double digestion. The ligation product was transformed into E.coli DH5α competent cells and cultured in a medium containing ampicillin. The recombinant cloned plasmid identified by PCR and sequencing was named AcaNAP10 / pICET32. The recombinant expression plasmid was transformed into Escherichia col...

Embodiment 3

[0024] Example 3 Expression of serine protease inhibitor AduKuI4 and its separation and purification

[0025]AduKuI4 is a Kunitz-type serine protease inhibitor isolated from Ancylostoma duodenale by the inventor's research group. It contains 3 pairs of disulfide bonds. The amino acid sequence of its mature peptide is: RNPHRKGRCGDDPAETGGECPDPETKYTYKFGDCHEVKYCGEQETRNLFDSYEKCSGKCVIF. According to the mature peptide coding sequence of AduKuI4, the upstream primer: 5'-TAGGATCCCGCAATCCTCACAGAAAG-3' and the downstream primer: 5'-CCAAGCTTAGAAGATCACGCACTTTCC-3' were designed, and the mature peptide coding sequence was amplified from the adult cDNA of Ancylostoma duodenale. The amplified product was ligated into the expression vector to successfully construct the pICET32 / AduKuI4 expression plasmid. The plasmid was transformed into Escherichia coli BL21(DE3), induced and cultured, the cells were collected by centrifugation, and the supernatant was collected after sonication, and added wi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention relates to a prokaryotic expression vector and application thereof. The vector comprises: (a), a molecular chaperone for promoting the soluble expression of target protein or / and the correct folding of disulfide bonds; and (b), an encoding nucleotide sequence of intein which can be broken on peptide bonds at a C terminal. In the vector, the high-efficiency soluble expression of the target protein is promoted by the molecular chaperone, so that the target protein maintain natural bioactivity, and the self-shearing action of the intein is induced effectively under a certain condition to separate the target protein from fusion protein, and the target protein is separated and purified again to obtain the target protein with natural bioactivity economically and efficiently. The invention also relates to host cells containing the vector and application of the vector in the recombinant expression of bioactivity protein.

Description

technical field [0001] The invention belongs to the technical field of bioengineering. Specifically, the present invention relates to a prokaryotic expression vector, a host cell containing the vector and the application of the vector in recombinantly expressing biologically active proteins. technical background [0002] Escherichia coli is currently the main engineering bacteria for the production of recombinant foreign proteins. However, prokaryotic systems are currently used to express and produce recombinant proteins, and recombinant proteins often appear in the form of insoluble inclusion bodies, low expression levels, and low biological activity. Linking molecular chaperone (molecular chaperone or chaperone; also called fusion partner, fusion partner or fusion tag, fusion tag) and target protein into a fusion protein for expression is one of the common strategies to increase the soluble expression of target protein. The most commonly used molecular chaperones are glu...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/16C12R1/19C12N15/74C12N1/21C12P21/02C12P21/00C12N15/70C07K1/22
Inventor 彭礼飞杨陈殷环邓莉甘伟琼廖淑莉
Owner GUANGDONG MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com