Preparation and preservation method for colon bacillus acetohydroxyacid synthase

A technology of acetolactate synthase and Escherichia coli, applied in the field of genetic engineering, can solve the problems of poor enzyme stability, complicated operation and low AHAS protein content, and achieve the effects of good stability, high purity and good activity

Inactive Publication Date: 2014-04-09
NORTHWEST UNIV
View PDF3 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of preparing recombinant AHAS by genetic engineering technology has also been reported. Most of the vectors used are pET series vectors, but the amount of expressed AHAS protein is relatively low, or it is expressed in the form of inclusion bodies, and it needs to go through steps such as denaturation and renaturation. The operation is complicated and the cost is high,

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
  • Preparation and preservation method for colon bacillus acetohydroxyacid synthase
  • Preparation and preservation method for colon bacillus acetohydroxyacid synthase
  • Preparation and preservation method for colon bacillus acetohydroxyacid synthase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0056] Main materials used in this embodiment:

[0057]

[0058]

[0059] Preparation of media and reagents:

[0060] (1) LB (Luria-Bertani) liquid medium: Weigh 5g yeast extract, 10g tryptone, 10g NaCl, dissolve in 800mL double distilled water (ddH 2 O), then adjust the pH to 7.0 with 2M NaOH, and then use ddH 2 O diluted to 1L, autoclaved at 121°C for 20min, and stored at 4°C for use; (2) LB solid medium: weigh 0.5g yeast extract, 1g tryptone, 1g NaCl, 1.5g agar, and dissolve in 90mL wxya 2 O, then adjust the pH to 7.0 with 2M NaOH, and then use ddH 2 Dilute O to 100mL, autoclave at 121°C for 20min, wait for the liquid to cool to about 60°C, pour it into 6 sterilized petri dishes on average, and store it at 4°C after cooling for later use;

[0061] (3) Ampicillin (Amp) solution: weigh 0.1g of Amp, dissolve it in 1mL of sterile water to make the final concentration 100mg / mL, and store it at -20°C after aliquoting;

[0062] (4) Isopropyl-β-D-thiogalactopyranoside (I...

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 provides a method for preparing AHAS (Acetohydroxyacid Synthase) which has good purity and high activity and can exist stably. The method comprises the following steps: upstream and downstream primers are designed according to a gene sequence of a colon bacillus AHAS catalytic subunit, wherein the designed upstream primer carries an Xho I enzyme digestion site and the downstream primer carries a BamHI enzyme digestion site; a colon bacillus BL21 strain genomic DNA (Deoxyribonucleic Acid) is used as a template and a target segment ahas is obtained by a PCR (Polymerase Chain Reaction) amplification technology; the target segment ahas is connected onto an expression carrier PGEX-AT-1 to obtain a recombinant plasmid PGEX-4T-1-ahas; inducible expression is realized in a prokaryotic expression system; an expressed enzyme is purified by agarose gel resin modified by glutathione sulfur transferase (GST) so as to obtain the acetohydroxyacid synthase with good activity.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to methods for cloning, soluble expression, purification and preservation of Escherichia coli acetolactate synthase gene. Background technique [0002] Acetolactate synthase (acetolactate synthase, AHAS, EC4.1.3.18) was first discovered in Escherichia coli (E.coli). So far, AHAS genes derived from 60 different biological species can be searched in GenBank, and these organisms are mainly bacteria, algae, fungi, etc. Usually, there may be many genes with high homology with AHAS gene in an organism, but not every gene can play a corresponding role with AHAS, and the AHAS gene in different parts of the same organism There are also large differences in copy number. In some lower organisms, the nucleotide sequence of AHAS is highly conserved, and its similarity is generally about 80%. The AHAS of Escherichia coli is a tetramer consisting of two large subunits and two smal...

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
IPC IPC(8): C12N9/88C12R1/19
CPCC12N9/1022C12N15/70C12Y202/01006
Inventor 高文运李恒刘楠王文婷
Owner NORTHWEST UNIV
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