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

Method for removing recombinant protein endotoxin by GEM

A recombinant protein and endotoxin technology, applied in biochemical equipment and methods, enzymes, hydrolases, etc., can solve problems such as destroying protein activity, and achieve the effect of reducing the loss of protein activity

Inactive Publication Date: 2020-02-11
TIANJIN UNIV
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for removing recombinant protein endotoxin by using GEM, which solves the problem in the prior art that heating and cooling are required to remove endotoxin, and repeated heating and cooling may destroy the activity of the 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
  • Method for removing recombinant protein endotoxin by GEM
  • Method for removing recombinant protein endotoxin by GEM
  • Method for removing recombinant protein endotoxin by GEM

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: Construction and expression of L-asparaginase-ACMA

[0024] 1) Vector construction

[0025] Using the Escherichia coli AS 1.357 genome as a template, the L-asparaginase gene was amplified and connected to the plasmid pET28a-AcmA to obtain the recombinant expression plasmid pET28a-LA-AcmA, which was transformed into the Escherichia coli expression vector BL21 by chemical transformation.

[0026] 2) Induced expression

[0027] Isolate by streaking to obtain a single clone of BL21 carrying the plasmid, culture overnight to obtain a seed solution, inoculate with an inoculum amount of 1%-5%, when the OD of the bacterial solution 600 When it reaches 0.5-0.8, add IPTG for induction, the induction concentration is 0.1mM-1mM, the induction temperature is 25°C-37°C, the induction time is 12 hours-24 hours, and all media are supplemented with 50μg / mL kanamycin LB medium. Using SDS-PAGE to analyze the recombinant expression, the results are as follows figure 1 As ...

Embodiment 2

[0028] Embodiment 2: GEM purification immobilized recombinant L-asparaginase

[0029] 1) GEM preparation

[0030] Cultivate Lactococcus lactis NZ9000 to OD in M17 medium containing 1% glucose 600 Between 2.0-2.5, collect the bacteria by centrifugation at 4000rpm, wash with PBS to remove the medium, resuspend the bacteria in 0.1M-0.2M HCl, boil for 30-60 minutes, collect the precipitate by centrifugation at 8000rpm, use 50mM Tri- HCl (pH 7.2) was washed thoroughly to obtain GEM.

[0031] 2) GEM purification of immobilized recombinant L-asparaginase-AcmA

[0032] Collect the induced bacteria by centrifugation at 4000rpm, wash and remove the culture medium with pre-cooled 50mM Tri-HCl (pH7.2), resuspend the bacteria in pre-cooled 50mM Tri-HCl (pH7.2), and sonicate Cells, sonicate for 2 seconds, pause for 4 seconds, sonicate for 20 minutes, centrifuge at 8000rmp for 20 minutes to separate, take the supernatant and mix with the GEM prepared in step 3), and combine with magnetic ...

Embodiment 3

[0034] Example 3: Endotoxin removal

[0035] The immobilized recombinant L-asparaginase-ACMA was resuspended in 1% Triton X-114 (4°C), combined on ice for 30 minutes, centrifuged at 8000 rpm for 10 minutes (4°C), the supernatant was discarded, and washed four times. Using SDS-PAGE for analysis, the results are as follows image 3 as shown in a. Determination of the enzyme activity after four rounds of removal, the results are as follows image 3 As shown in b, more than 80% of the relative enzyme activity was still preserved after four rounds of clearance work.

[0036] In this embodiment, 1% Triton X-114 is used to remove endotoxin at low temperature (4° C.), which can guarantee protease activity.

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 discloses a method for removing recombinant protein endotoxin by a GEM, and relates to a method for removing endotoxin in an escherichia coli expression recombinant protein. After a protein-AcmA fusion protein recombination expression carrier is subjected to inducible expression, the GEM is used for carrying out purification and immobilization, and then, Triton X-114 is used for processing the immobilized protein to remove the endotoxin. By use of the method, endotoxin removal work is carried out at a low temperature of 4 DEG C, a situation that the activity of the protein is affected since a temperature is changed is avoided, and an endotoxin removal operation is simplified.

Description

technical field [0001] The invention relates to a method for removing recombinant protein endotoxin by using GEM. Background technique [0002] Proteins can be used as enzymes for catalysis, and can also be used as drugs for disease treatment. The natural protein produced by wild bacteria has the disadvantages of low yield and complicated purification methods. These shortcomings can be improved by using recombinant expression. Induction with IPTG can achieve overexpression, greatly improve the yield, and simplify purification by adding tags. [0003] Bacterial endotoxin (endotoxin), also known as lipopolysaccharide (LPS), is one of the main components of the outer membrane of the cell wall of Gram-negative bacteria. In recombinant expression of Escherichia coli, endotoxins in the cell wall are also released into the protein solution in large quantities when the bacteria are broken to obtain proteins. The presence of endotoxins can cause strong inflammatory reactions in an...

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): C12N9/82
CPCC12N9/82C12Y305/01001
Inventor 韩烨赵芳坤肖华志周志江
Owner TIANJIN 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