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

Production method for protein

A technology of protein and control region, applied in botany equipment and methods, biochemical equipment and methods, using vectors to introduce foreign genetic material, etc., can solve problems such as finding no efficient method for high expression of Cry protein

Pending Publication Date: 2020-08-28
KAO CORP
View PDF12 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, it has also been reported that in Streptococcus lactis, when a high copy number plasmid is used to express the Cry5B protein gene under the control of a high expression promoter, the productivity of the protein of Bacillus thuringiensis can only be detected at the immunoblot level (Non-Patent Document 9 )
[0005] In this way, methods for generally increasing the expression of proteins outside cells have been studied, but it cannot be said that they can be applied to the production of proteins inside cells. In particular, an efficient method for high expression of Cry proteins has not yet been found.

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
  • Production method for protein
  • Production method for protein
  • Production method for protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Example 1 Production of Cultures Containing Cry5B Protein

[0135] 1. Synthesis of artificial genes

[0136]The insecticidal protein gene cry5B (GenBank: CP005935.1, sequence number 1) from Bacillus thuringiensis YBT-1518 was artificially synthesized by GenScript Company (USA). The 3738bp of the synthesized gene was cloned into the KpnI and HindIII sites of pUC57 to obtain pUC57-cry5B.

[0137] 2. Construction of expression plasmids

[0138] In constructing all the expression plasmids, pHY300PLK (TAKARA BIO) was used as the vector, and the sequence derived from the S237 cellulase gene was used as the terminator. Promoter from S237 cellulase gene [Hakamada et al, Biosci.Biotechnol.Biochem., 64(2000), 2281-2289] or spoVG gene of Bacillus subtilis strain 168, signal sequence from S237 cellulase or none, cry5B gene By combining full-length (cry5B) or shortened (cry5Bt), 8 kinds of plasmids ( Figure 1-A ~ D). The full length (3738bp) of the cry5B gene is represented by ...

Embodiment 2

[0199] Embodiment 2 Production of mosquito-killing protein (Cry4Aa, Cry4Ba, Cry11Aa)

[0200] 1. Synthesis of artificial genes

[0201] The insecticidal protein genes cry4Aa (GenBank: YP_001573833, sequence number 3), cry4Ba (GenBank: NC_010076, sequence number 5) and cry11Aa (GenBank: NC_010076, sequence number 5) and cry11Aa ( GenBank: NC_010076, Sequence No. 7). The synthesized genes were cloned into the KpnI and HindIII sites of pUC57 to obtain plasmids pUC57-cry4Aa, pUC57-cry4Ba and pUC57-cry11Aa, respectively.

[0202] 2. Construction of expression plasmids

[0203] In constructing all the expression plasmids, pHY300PLK was used as the vector, and the sequence derived from the S237 cellulase gene was used as the terminator. The promoter is passed through the S237 cellulase gene [Hakamada et al, Biosci.Biotechnol.Biochem., 64(2000), 2281-2289] or the spoVG gene from Bacillus subtilis 168 strain, cry4Aa, cry4Ba , cry11Aa combination of 3 genes, carry out Fi...

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

Provided is a production method for high production of crystal protein inside bacteria cells of Bacillus bacteria. This production method is for crystal protein or a cultured product containing same and includes: transforming Bacillus bacteria using an expression plasmid wherein a gene that codes crystal protein and a control area including a sigma A-dependent promoter or a sigma H-dependent promoter are operably linked and incorporated; and said transformed cells are cultured. The expression plasmid includes a polynucleotide that codes a replication protein comprising an amino acid sequence expressed by SEQ ID NO: 9 or a protein having at least 80% homology with same in the amino acid sequence and is involved to replication initiation.

Description

technical field [0001] The present invention relates to a method for producing a Cry protein using bacteria belonging to the genus Bacillus. Background technique [0002] In general, Escherichia coli is mostly used, and when a large amount of protein is expressed in the bacteria, it is recovered as an insoluble and inactivated aggregate (modified protein) called an inclusion body (inclusion body). Bioactivity requires operations of solubilization and activation (refolding) of aggregates (Non-Patent Document 1). Even so, if no active protein can be obtained, the expression level is adjusted so that inclusion bodies are not formed (Non-Patent Documents 2 and 3). In addition, when producing heterologous proteins in cells, it is necessary to adjust the expression of promoters and the number of plasmid copies due to the influence of heterologous proteins on the host, etc. (Non-Patent Documents 2 and 3). [0003] On the other hand, in the expression of proteins outside bacteria,...

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): C12N15/32C12N1/21C12N15/75C12P21/02
CPCC12N15/75C07K14/325C07K14/32
Inventor 刘生浩影山泰寺井三佳
Owner KAO CORP
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