Protein synthesis system for increasing expression amount of foreign protein and application method of protein synthesis system

A synthetic system and protein synthesis technology, applied in the field of bioengineering, can solve the problems of low and unsatisfactory protein translation and synthesis

Inactive Publication Date: 2020-03-31
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the efficiency of protein translation and synthesis of the current in vitro biosynthesis system in this field is still low and unsatisfactory. Therefore, there is an urgent need in this field to develop a new system and method that can improve the efficiency of foreign protein translation and synthesis

Method used

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  • Protein synthesis system for increasing expression amount of foreign protein and application method of protein synthesis system
  • Protein synthesis system for increasing expression amount of foreign protein and application method of protein synthesis system

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preparation example Construction

[0193] In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0194] (i) providing yeast cells;

[0195] (ii) washing the yeast cells to obtain washed yeast cells;

[0196] (iii) subjecting the washed yeast cells to destructive treatment to obtain crude yeast extract;

[0197] (iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

[0198] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0199] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0200] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000×g, preferably 8000-30000×g.

[0201] In the present invention, the centrifugation time is not parti...

Embodiment 1

[0217] Example 1 Inserting a strong promoter before the KlEAP1 gene by CRISPR-Cas9 technology

[0218] 1.1 KlURA3 sequence search and CRISPR gRNA sequence determination

[0219] URA3 gene is a gene on chromosome V of yeast, which can carry out positive selection and negative selection, and is one of the most important genetic markers in yeast genetic engineering. In the embodiment, URA3 is selected as the screening marker, and of course other commonly used screening markers (genetic markers) can also be selected.

[0220] Based on the URA3 gene sequence in Saccharomyces cerevisiae, the URA3 gene sequence was compared and analyzed in the UniProt database to determine the URA3 homologous gene sequence in Kluyveromyces lactis, named KlURA3 (located at 2034995..2035798 on chromosome E ). At the stop codon of KlURA3 gene, the adjacent PAM sequence (NGG) was searched, and the gRNA sequence was determined. The principle of gRNA selection is: moderate GC content, 40%-60%; avoid the...

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Abstract

The invention provides a protein synthesis system for increasing the expression amount of a foreign protein and an application method of the protein synthesis system. Specifically, a poly-nucleotide sequence or vector constructed by a strong promoter sequence is inserted in front of a nucleotide sequence for encoding an eIF4E binding protein, over-expression of eIF4E binding proteins such as Eap1and p20 can be achieved, and foreign protein synthesis is implemented by using the improved cell lysis buffer. The invention provides the protein synthesis system for increasing the expression amountof the foreign protein, and by adopting the synthesis system provided by the invention, the efficiency of protein translation can be remarkably improved.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a protein synthesis system for increasing the expression of exogenous proteins and an application method thereof. 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 cells, the regulation of protein translation plays an important role in many processes such as response to external stress such as nutrient deficiency, cell development and differentiation. The four processes of protein translation include translation initiation, translation elongation, translation termination, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/67
CPCC12N15/67C12N15/81C12N15/815
Inventor 郭敏丁晓辉刘显成杨宁王绍杰董颖颖王静代田纯于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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