Quick clone and expression plasmid vector containing N utilization substance A (NusA) protein fusion label

A plasmid carrier and protein expression technology, applied in the field of molecular biology, can solve the problems of expensive proteases and limited fusion tags, and achieve the effects of separation and purification, fast and efficient cloning and protein expression, and saving experimental costs

Inactive Publication Date: 2013-01-16
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
View PDF1 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the commercial carriers currently on the market can use very limited fusion tags, and the protease designed on the carrier to separate the target protein from the tag is also relatively expensive.

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
  • Quick clone and expression plasmid vector containing N utilization substance A (NusA) protein fusion label
  • Quick clone and expression plasmid vector containing N utilization substance A (NusA) protein fusion label
  • Quick clone and expression plasmid vector containing N utilization substance A (NusA) protein fusion label

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0016] The vector of the present invention uses pET-24d as the starting plasmid, and during the construction process, the gene sequence for encoding NusA protein, TEV restriction site and GFP gene are respectively added. A BsaI single restriction site was introduced on both sides of the GFP gene. On the one hand, the vector is linearized by BsaI single enzyme digestion and 4 base sticky ends protruding from the 5' end are produced on both sides of the linearized fragment. After being treated with T4DNAPolymerase and dTTP, the number of bases at the sticky ends Each increased to 10 and 12, using the cohesive ends, can conveniently and quickly clone the target gene into the vector and perform protein expression and purification. On the other hand, in order to control the quality of the clone faster and more conveniently, the GFP gene inserted in the middle of the restriction site BsaI site of the clone can be judged by observing whether it produces green fluorescence during the ...

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

No PUM Login to view more

Abstract

The invention relates to a clone and expression plasmid vector. The clone and expression plasmid vector contains a green fluorescent protein (GFP) gene segment which contains a tobacco etch virus (TEV) enzyme recognition site and is used for encoding the gene sequence of NusA protein, and of which two sides are provided with bovine serum albumin (BsaI) restriction enzyme cutting sites. The constructed vector can realize the quick and efficient clone and protein expression of the targeted gene without depending on ligase. The NusA protein label and the TEV enzyme recognition site are introduced into the vector; and by the NusA protein label, the solubility of the insoluble protein can be improved, and the protein can be separated and purified conveniently; and a TEV enzyme recognition sequence is positioned at the downstream of the gene sequence of the NusA protein label, the purified protein is cut by TEV enzyme, and the NusA protein label can be cut off, so that the natural targeted protein can be obtained. The TEV enzyme is the enzyme which is the most economic and applicable at present, so that the experimental cost is greatly saved.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a cloning and protein expression plasmid carrier, that is, a rapid cloning and protein expression plasmid carrier containing a NusA protein fusion tag. Background technique [0002] The traditional cloning method is to generate sticky ends of 2-4 bases by digesting the plasmid vector and the target gene fragment respectively with the same double enzyme digestion, and then use ligase to connect the complementary sticky ends, and then transform Escherichia coli cells, Express purified protein. Due to the relatively short length of the cohesive end, T4 DNA ligase must be used to ligate the vector and the target gene fragment, and some vectors may self-ligate during this process. This method takes about two to three days to clone a gene. In order to realize a ligase-independent cloning method, it is necessary to construct a more convenient and effective clonin...

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): C12N15/70
Inventor 邹培建盖园明王鹏举景小飞
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap