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In-vitro cell-free protein synthesis system (D2P system) and kit and use thereof

A cell-free protein synthesis system technology, applied in the field of in vitro cell-free protein synthesis system optimized by L-arabinose, to achieve cost saving, increase protein synthesis yield, and simple operation

Pending Publication Date: 2021-08-06
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In summary, how to improve the in vitro cell-free protein synthesis system based on Kluyveromyces lactis, there are still many technical gaps to be solved

Method used

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  • In-vitro cell-free protein synthesis system (D2P system) and kit and use thereof
  • In-vitro cell-free protein synthesis system (D2P system) and kit and use thereof
  • In-vitro cell-free protein synthesis system (D2P system) and kit and use thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0193] The preparation method of the cell extract can adopt the reported technical means. In a brief summary, it usually includes the following steps: quick-freeze the cells with liquid nitrogen, break up the cells, and collect the supernatant by centrifugation to obtain the cell extract. Reference CN106978349A, CN108535489A, CN108642076A, CN109593656A, CN109971783A and other documents. The seed cells can be fermented and cultured, centrifuged, the culture medium is removed, the cells are collected, and then the cell extract is prepared.

[0194] The cell extract prepared by the method provided by the invention can make the in vitro protein synthesis reaction go on normally, and contains necessary components required for protein synthesis such as tRNA with amino acid transport function and aminoacyl tRNA synthetase.

[0195] In one embodiment, the cell extract is prepared by a method comprising the following steps: (i) providing source cells, Kluyveromyces lactis cells; (ii) ...

Embodiment 1

[0329] Example 1 Effect of the concentration of L-arabinose on the protein synthesis ability of the in vitro protein synthesis system

[0330] 1.1 Preparation of nucleic acid template: construct a plasmid vector expressing mEGFP, carry out in vitro DNA amplification, and prepare a DNA template (plasmid DNA template) encoding exogenous protein mEGFP

[0331] Enhanced green fluorescent protein (mEGFP) was selected as the foreign protein as the target expression product, and its amino acid sequence was shown in SEQ ID No.2.

[0332] Select a plasmid vector. The artificially constructed plasmid vector designed for the Kluyveromyces lactis cell extract contains functional elements such as T7 promoter, 5'UTR and 3'UTR. The plasmid vector can be combined with the Kluyveromyces lactis cell extract containing endogenously expressed T7 RNA polymerase to construct an in vitro cell-free protein synthesis system to express various exogenous proteins in vitro.

[0333] Using PCR amplifica...

Embodiment 2

[0350] Example 2 Effect of the concentration of L-arabinose on the protein synthesis ability of the in vitro protein synthesis system

[0351] 2.1 Preparation of nucleic acid template: The method of 1.1 in Example 1 was used to construct a plasmid vector expressing mEGFP and perform in vitro DNA amplification to prepare a plasmid DNA template encoding exogenous protein mEGFP.

[0352] 2.2 Referring to the method of 1.2 in Example 1, based on the Kluyveromyces lactis strain ATCC8585, the coding gene of T7 RNA polymerase was integrated into the genome of Kluyveromyces lactis to obtain the modification of endogenously expressing T7 RNA polymerase strain. After fermenting and cultivating the modified strain, the Kluyveromyces lactis cell extract is prepared, and the number is YY09161.

[0353] 2.3 In vitro cell-free protein synthesis reaction system (without adding exogenous RNA polymerase)

[0354] The volume of the reaction system was 300 μL, and the reaction was carried out i...

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Abstract

The invention provides an in-vitro cell-free protein synthesis system and a kit and use thereof, and belongs to the technical field of protein synthesis. The in-vitro cell-free protein synthesis system based on a kluyveromyces lactis source is improved by adding exogenous L-arabinose, such that the protein synthesis capability of the system is remarkably improved. The invention further provides a more efficient and higher-throughput in-vitro protein synthesis kit and a synthesis method of a foreign protein. The provided improved method can be realized without molecular modification, is simple and convenient to operate and saves cost.

Description

technical field [0001] The present invention relates to the technical field of protein synthesis, in particular to the technical field of in vitro cell-free protein synthesis, in particular to an L-arabinose-optimized in vitro cell-free protein synthesis system (DNA-to-Protein system, D2P system), its kit and its application. Background technique [0002] Proteins are important molecules in cells and are involved in almost all functions of cells. Protein synthesis mainly includes traditional intracellular synthesis technology and new generation in vitro synthesis technology. The traditional protein expression system refers to a molecular biology technique that expresses foreign genes through model organisms such as bacteria, fungi, plant cells, insect cells or animal cells. The in vitro protein synthesis system, also known as the cell-free expression system, came into being in the 1960s. It uses exogenous mRNA or DNA as a template for protein synthesis, and artificially co...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12P21/00C12R1/645
CPCC12N1/16C12P21/00
Inventor 郭敏章小铃于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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