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Novel fusion protein preparation method and application of novel fusion protein for increasing protein synthesis

A protein synthesis and fusion protein technology, which is applied in the field of improving protein synthesis and the preparation of new fusion proteins, can solve the problems of low efficiency, slow speed, and limitation of protein synthesis applications, and achieve the effect of improving translation efficiency

Active Publication Date: 2018-10-23
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, both intracellular and extracellular artificial protein synthesis systems in nature have the characteristics of low efficiency and slow speed, which greatly limits the application of protein synthesis.

Method used

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  • Novel fusion protein preparation method and application of novel fusion protein for increasing protein synthesis
  • Novel fusion protein preparation method and application of novel fusion protein for increasing protein synthesis
  • Novel fusion protein preparation method and application of novel fusion protein for increasing protein synthesis

Examples

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preparation example Construction

[0141] In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0142] (i) providing yeast cells;

[0143] (ii) washing the yeast cells to obtain washed yeast cells;

[0144] (iii) subjecting the washed yeast cells to destructive treatment to obtain crude yeast extract;

[0145] (iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

[0146] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0147] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0148] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000×g, preferably 8000-30000×g.

[0149] In the present invention, the centrifugation time is not parti...

Embodiment 1

[0193] Example 1 Theoretical Model for Improving Protein Synthesis Through Genetic Modification

[0194] The present invention uses CRISPR-Cas9 gene editing technology to K. lactis The translation initiation factors eIF4G and Pab1 were optimized to improve the efficiency of the cell-free in vitro translation system.

Embodiment 2

[0195] Example 2 Transformation of translation initiation factors by CRISPR-Cas9 to improve the efficiency of in vitro translation systems

[0196] 2.1 Translation initiation factor by CRISPR-Cas9 technology ikB strong promoter

[0197] 2.1.1 KleIF4G sequence search and CRISPR gRNA sequence determination

[0198] eIF4G is an important factor in the process of translation initiation. In the current reports, there is no case of optimizing the expression of endogenous eIF4G through gene editing technology to improve in vitro translation activity. According to the theoretical model in Example 1, the present invention uses the CRISPR-Cas9 gene editing technology to modify the expression of the translation initiation factor KleIF4G, so as to improve the efficiency of the cell-free in vitro translation system.

[0199] based on S. cerevisiae in yeast eIF4G gene sequence. in the NCBI database as eIF4G Gene comparison analysis by BLAST, to determine the Kluyveromyces lactis ...

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Abstract

The invention provides a novel fusion protein preparation method and an application of the novel fusion protein for increasing protein synthesis. The provided fusion protein can greatly increase the in-vitro translation efficiency. In addition, a composition-type or induction-type promoter (such as pK1PGK1) is inserted before eIF4G, and the synthesis capability of the in-vitro protein is obviouslyenhanced.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the preparation of a novel fusion protein and its application in improving protein synthesis. 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. [0003] 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 pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/14C12N15/62C12P21/00C12N9/02
CPCC12N9/0069C12N9/14C12P21/00C12Y113/12007C12Y306/04013C07K2319/02C07K2319/00C12P21/02C07K14/39C12N15/62C07K7/06C12N15/86
Inventor 郭敏代田纯王海鹏薛银鸽柴智刘帅龙于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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