A method and expression system for cell-free expression of signaling protein

An expression system and cell-free technology, which is applied in the field of cell-free expression of signal proteins, methods and expression systems, can solve the problem of low expression of signal proteins, achieve large protein expression, correct structure, and reduce the effect of foreign protein content

Active Publication Date: 2020-02-04
CUSABIO TECH LLC
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  • Abstract
  • Description
  • Claims
  • Application Information

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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 system for cell-free expression of signaling proteins, which can solve the problem of low expression of signaling proteins

Method used

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  • A method and expression system for cell-free expression of signaling protein
  • A method and expression system for cell-free expression of signaling protein
  • A method and expression system for cell-free expression of signaling protein

Examples

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Embodiment 1

[0067] Example 1: Take the porcine β2 adrenergic receptor as an example to perform the cell-free signal protein expression in the examples of this application.

[0068] 1) Construction of expression vector

[0069] Using the total RNA of porcine hepatocytes as a template, the RT-PCR reaction was performed referring to the operation steps of the RT-PCR kit. The RT-PCR product was detected by 1% agarose gel electrophoresis. A 50μL reaction system was used, as follows: 22μL water (nuclease-free), 10μL AMV / Tfi 5× reaction buffer, 1μL dNTP mixture (10mmol / L per dNTP), 5μL upstream primer (10pmol / μL), downstream Primer (10pmol / μL) 5μL, MgSO 4 (25mmol / L) 2μL, vortex gently to mix, add 1μL of AMV reverse transcriptase (5U / μL), 1μL of DNA polymerase (5U / μL), shake slightly for 10s and add 3μL of template RNA. RT-PCR reaction conditions: 50 ℃ 45 min, reverse transcription reaction; 94 ℃ 2 min, inactivation of reverse transcriptase and pre-denaturation; 94 ℃ denaturation 30 s, 59 ℃ anneali...

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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 expression system for cell-free expression of signal proteins. Background technique [0002] Most signal proteins are membrane proteins. How to efficiently express membrane proteins has always been the main bottleneck in the field of signal protein research. In most of the various genomes that have been sequenced, the number of membrane proteins only accounts for one-third of the total number of genes. There are only a limited number of membrane proteins whose spatial structure has been measured. The main reason is that the extremely high hydrophobicity of membrane proteins causes its expression in the cell body to produce toxicity to the host cell, resulting in low protein yield. Moreover, the insoluble expression of membrane proteins and the misfolding of proteins can lead to a reduction or even loss of biological activity. The in vivo expression of some membrane pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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