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

Prokaryotic recombinant expression and preparation method of lysyl endopeptidase

A technology of lysyl peptide chain and endopeptidase, which is applied in the field of genetic engineering and can solve the problems such as the renaturation of preL recombinantly expressed proteins.

Active Publication Date: 2016-09-21
ACADEMY OF MILITARY MEDICAL SCI
View PDF5 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, similar to Le-LEP, there is no successful report on the recombinant expression of preL based on prokaryotic expression system and protein renaturation.

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 recombinant expression and preparation method of lysyl endopeptidase
  • Prokaryotic recombinant expression and preparation method of lysyl endopeptidase
  • Prokaryotic recombinant expression and preparation method of lysyl endopeptidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1. Preparation and identification of recombinant lysyl peptide chain endopeptidase (Lys-C)

[0090] In this example, the E. coli prokaryotic expression system will be used to express recombinant lysyl peptide chain endopeptidase (Lys-C) and identify it.

[0091] 1. Preparation of recombinant E. coli expressing recombinant lysyl peptide chain endopeptidase (Lys-C)

[0092] 1. Optimization of Lysyl Peptidase (Lys-C) coding gene

[0093] The Lys-C encoding gene (GenBank number: AY062882.1, position 73-1389, GI: 17978564) derived from Pseudomonas aeruginosa, without changing the amino acid of the wild-type lysyl peptide chain Under the premise of the sequence (positions 169-606 of sequence 2), replace its codons with codons preferred by E. coli (high frequency use). The optimization also includes other modifications to the gene sequence of the peptidase within the lysyl peptide chain to be suitable for expression in E. coli, and the start codon ATG is added before the opti...

Embodiment 2

[0140] Example 2. Activity determination of recombinant lysyl peptide chain endopeptidase (Lys-C)

[0141] 1. Preparation of E. coli whole protein

[0142] E. coli BL21(DE3) (Novagen, Germany) was resuspended in PBS and then ultrasonically broken and centrifuged to obtain total bacterial protein. The protein was precipitated with pre-chilled acetone in a ratio of 1:9, and then resuspension buffer (formulation :The solvent is water; the solute and concentration are 50mM NH 4 HCO 3 And 8M urea) to resuspend, the protein concentration is 4mg / ml. Add DTT to a final concentration of 20 mM, and denature at 37°C for 45 min. After cooling the sample to room temperature, add iodized acetamide to a final concentration of 40 mM and carry out alkylation in the dark. The alkylation time is 30 min. After the alkylation is over, a 10kD ultrafiltration tube is used to change the liquid to remove DTT and acetamide iodide. Use buffer (formulation: solvent is water; solute and concentration are 50m...

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
Molecular weightaaaaaaaaaa
Molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention discloses a prokaryotic recombinant expression and preparation method of lysyl endopeptidase. The preparation method comprises the following steps: importing encoding genes of recombinant lysyl endopeptidase with the amino acid sequence 2 into recipient escherichia coli cells, thus obtaining recombinant escherichia coli cells; carrying out inducible expression on the recombinant escherichia coli cells, collecting and crushing the recombinant escherichia coli cells, thus obtaining an inclusion body; and sequentially carrying out denaturation, renaturation, activation and purification to obtain the recombinant lysyl endopeptidase. The recombinant lys-C protease prepared by adopting the preparation method provided by the invention has high enzyme activity and relatively high urea tolerance. The preparation method has the advantages that the enzyme specificity is good, no animal-origin viruses exist, etc.; the production and preparation processes are simple and are low in cost; and the method can be applied to biopharmacy and proteomic analysis.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and relates to a prokaryotic recombinant expression and preparation method of a lysyl peptide chain endopeptidase. Background technique [0002] Lysyl endopeptidase (EC 3.4.21.50) can specifically digest the lysine at the C-terminus of the protein substrate. There are three main types of Lysyl endopeptidase reported at present, namely Achromobacter protease I from Achromobacter lyticus (API), lysyl endopeptidase from Lysobacterenzymogenes and preL from Pseudomonas aeruginosa. Research on API (A-LEP) shows that API is expressed in the form of zymogen when expressed. Its complete sequence includes a signal peptide sequence of 20 amino acids, a pro-peptide of 185 amino acids at the N-terminal and 180 amino acids at the C-terminal. The extension peptide and the mature API composed of 268 amino acids. API has higher activity, wider pH tolerance and surfactant tolerance than trypsin. These characterist...

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): C12N9/52C12N15/57C12N15/70
Inventor 赵明治徐平吴飞林常蕾
Owner ACADEMY OF MILITARY MEDICAL SCI
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