Method for cell-free expression of signal protein and expression system

An expression system and cell-free technology, which is applied in the field of cell-free expression of signaling proteins, methods and expression systems, can solve the problem of low expression of signaling proteins, and achieve the effect of large protein expression, low price, and high concentration

Active Publication Date: 2016-10-12
CUSABIO TECH LLC
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Problems solved by technology

[0006] In view of the above problems, the main purpose of the present invention is to provide a method and expression sy

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  • Method for cell-free expression of signal protein and expression system
  • Method for cell-free expression of signal protein and expression system
  • Method for cell-free expression of signal protein and expression system

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Embodiment 1

[0067] Example 1: The expression of the cell-free signaling protein in the embodiment of the present application is carried out by taking the porcine β2 adrenergic receptor as an example.

[0068] 1), construction of expression vector

[0069] Using the total RNA of porcine hepatocytes as a template, RT-PCR reaction was carried out referring to the operation steps of the RT-PCR kit. RT-PCR products were detected by 1% agarose gel electrophoresis. A 50 μL reaction system was used, specifically as follows: 22 μL of water (nuclease-free), 10 μL of AMV / Tfi 5× reaction buffer, 1 μL of dNTP mixture (10 mmol / L per dNTP), 5 μL of upstream primer (10 pmol / μL), 5 μL of downstream Primer (10pmol / μL) 5μL, MgSO 4 (25mmol / L) 2μL, vortex slightly to mix, add AMV reverse transcriptase (5U / μL) 1μL, DNA polymerase (5U / μL) 1μL, shake slightly for 10s, then add template RNA 3μL. RT-PCR reaction conditions: 50°C for 45min, reverse transcription reaction; 94°C for 2min, reverse transcriptase i...

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Abstract

The invention relates to a method for cell-free expression of signal protein and an expression system. The method includes joining a target gene of the signal protein on a pIX3.0 vector to obtain expression plasmids; extracting escherichia coli Rosetta (DE3) by S30 buffer solution to obtain escherichia coli extract; preparing an energy supply system comprising PEP (phosphoenolpyruvic acid)/PK (pyruvate kinase), CP (phosphocreatine)/CK (creatine kinase) and glucose; preparing an amino acid mixture and a saline solution; preparing lecithin/cholesterol liposome; adding the expression plasmids into a cell-free protein expression system comprising the escherichia coli extract, the energy supply system, the amino acid mixture, the saline solution and the lecithin/cholesterol liposome for expression. The method is capable of solving the problem of small signal protein expression quantity.

Description

technical field [0001] The invention relates to the field of protein preparation, in particular to a method and an expression system for cell-free expression of signal proteins. Background technique [0002] Most of the signaling proteins are membrane proteins. How to efficiently express membrane proteins has always been the main bottleneck in the field of signaling protein research. In most genomes that have been sequenced, the number of membrane proteins accounts for only one-third of the total number of genes About ninety or so spatial structures of membrane proteins have been determined. The main reason is that the extremely high hydrophobicity of membrane proteins leads to toxicity to host cells when expressed in cells, resulting in low protein yields. Moreover, the insoluble expression of membrane proteins and the misfolding of proteins will lead to the reduction or even loss of biological activity. The in vivo expression of some membrane proteins will interfere with ...

Claims

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Application Information

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IPC IPC(8): C12N15/70
CPCC12N15/70C12N2800/101
Inventor 沈鹤霄华权高王静马峰徐春雷
Owner CUSABIO TECH LLC
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