Method for improving protein synthesis efficiency in cells

A protein synthesis, cell-free protein technology, applied in the field of improving protein synthesis efficiency, can solve the problems of leakage, lack of enzyme-enzyme complexes and cofactors, poor transcriptional modularity, etc., and achieve the effect of improving translation efficiency

Active Publication Date: 2019-08-06
KANGMA SHANGHAI BIOTECH LTD
View PDF3 Cites 28 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this system is not suitable for large-scale application due to adenosine triphosphate (ATP) imbalance [6]
Welch and Scopes solved the above problems through various explorations in 1985, and obtained high-yield ethanol, but this system also has two major defects: it needs to add high-cost enzymes and cannot tolerate temperature changes[7]
However, there are some inherent and difficult problems in this technology at present: such as reversibility, instability, leakage, inactivation, recycling of enzymes, lack of stable enzymes, enzyme complexes and cofactors, etc.
For example, only one sigma factor is present in cell-free extracts and transcriptional modularity remains poor

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 improving protein synthesis efficiency in cells
  • Method for improving protein synthesis efficiency in cells
  • Method for improving protein synthesis efficiency in cells

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0337] In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0338] (i) providing yeast cells;

[0339] (ii) washing the yeast cells to obtain washed yeast cells;

[0340] (iii) subjecting the washed yeast cells to destructive treatment to obtain crude yeast extract;

[0341] (iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

[0342] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0343] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0344] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000 g, preferably 8000-30000 g.

[0345] In the present invention, the centrifugation time is not parti...

Embodiment 1

[0364] Example 1 Construction of a new type of fusion protein, greatly improving in vitro translation efficiency

[0365] 1.1 Through CRISPR-Cas9 gene editing technology, optimize the translation initiation factors eIF4G and Pab1 in K. lactis, and fuse KleIF4G with its interacting protein to improve the efficiency of the cell-free in vitro translation system.

[0366] Sequence search and CRISPR gRNA sequence determination

[0367] The present invention uses CRISPR-Cas9 gene editing technology to fuse KlPab1 and KleIF4G to promote the interaction between the two, so as to improve in vitro translation efficiency.

[0368] Based on the Pab1 sequence, the KlPab1 gene sequence (located at 1553322...1555100 on chromosome C) in Kluyveromyces lactis was obtained. Search for the PAM sequence (NGG) near the stop codon of the KlPab1 gene, and determine the gRNA sequence. The principle of gRNA selection is: moderate GC content, the standard of the present invention is 40%-60% GC content...

Embodiment 2

[0381] Example 2 By knocking out the nuclease KlEXN53, endogenously expressing T7RNP with a specific activation strength ScRNR2, reducing or not adding additional T7RNP to improve in vitro translation efficiency.

[0382] Background and Analysis of Biosynthesis in Organisms

[0383] In vivo biosynthesis refers to the synthesis process of various compounds catalyzed by enzymes in organisms, including photosynthesis, gluconeogenesis, and biosynthesis of macromolecules such as nucleotides, nucleic acids, and proteins. Among them, protein synthesis is the most important in quantity.

[0384] The whole process of biosynthesis includes transcription and translation, which is the transfer of genetic information from DNA to RNA. That is, one strand of double-stranded DNA is used as a template, and four kinds of nucleoside triphosphates (adenotriphosphorus (ATP), cytotriphosphorus (CTP), guaninetriphosphorus (GTP) and urinary triphosphate (UTP)) are used as raw materials. The process...

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 improving protein synthesis efficiency in cells. Specifically, the present invention provides an engineering bacterial strain for in-vitro cell-free protein synthesis, a first exogenous gene expression cassette of a first nucleic acid construct expressing a first fusion protein is integrated into a genome of the gene engineering bacterial strain, in the gene engineering bacterial strain, the expression or activity of a KlEXN53 gene (nuclease gene) is lowered. A cell extract (such as a yeast cell extract) derived from the engineering bacterial strain of the invention can significantly increase the stability of nucleic acid without the need for additional manual addition of T7 RNP, and the production efficiency of protein in an in-vitro protein synthesis system can be significantly increase.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for improving protein synthesis efficiency in cells. Background technique [0002] Proteins are important molecules in cells and are involved in almost all functions of cells. Different sequences and structures of proteins determine their different functions. In cells, proteins can be used as enzymes to catalyze various biochemical reactions, and as signal molecules to coordinate various activities of organisms, to support biological forms, store energy, transport molecules, and make organisms move. In the field of biomedicine, protein antibodies, as targeted drugs, are an important means of treating diseases such as cancer [1-2]. [0003] In cells, protein production involves two parts: gene transcription and mRNA translation. [0004] Gene transcription refers to the process of using a strand of DNA as a template, under the catalysis of DNA-dependent RNA polymerase, and...

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): C12N1/19C12N15/81C12N15/67C12P21/02
CPCC12N15/815C12N15/67C12P21/02Y02E50/10
Inventor 郭敏李海洋代田纯姜灵轩章小铃刘帅龙于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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