Optimized in vitro cell-free protein synthesis system and application

A cell-free protein synthesis system technology, applied in the field of cell-free protein synthesis system, can solve the problems of high cost and insufficient reaction efficiency

Active Publication Date: 2020-07-07
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It mainly solves the technical problems of high cost and low reaction efficiency of the protein synthesis system in the prior art

Method used

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  • Optimized in vitro cell-free protein synthesis system and application
  • Optimized in vitro cell-free protein synthesis system and application
  • Optimized in vitro cell-free protein synthesis system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 In vitro cell-free protein synthesis system containing glucose + maltodextrin

[0073] In vitro protein synthesis reaction system: final concentration of 22 mM 4-hydroxyethylpiperazineethanesulfonic acid (Hepes-KOH) at pH 7.4, 120 mM potassium acetate, 5.0 mM magnesium acetate, 1.5 mM nucleoside triphosphate mixture (adenoside Purine nucleoside triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine triphosphate), 0.1 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine amino acid, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine), 1.7 mM dithiothreitol, 20 mM glucose, 20 mM tripotassium phosphate, 0-500 mM maltodextrin (measured as glucose monomer), 0.002 mg / mL of α-amylase, 0.03 mg / mL T7 RNA polymerase, 2% polyethylene glycol, 50% volume yeast cell extract, 15 ng / µL enhanced green fluorescent protein DNA...

Embodiment 2

[0079] Example 2 Optimization of in vitro cell-free protein synthesis system

[0080] 2.1 Original in vitro cell-free protein synthesis system (system 2): 4-hydroxyethylpiperazineethanesulfonic acid (Hepes-KOH) at a final concentration of 22 mM pH 7.4, 120 mM potassium acetate, 5 mM magnesium acetate, 1.5 mM commercial nucleoside triphosphate mixture liquid (adenosine triphosphate, guanosine triphosphate, cytidine nucleoside triphosphate and uridine triphosphate), 0.1 mM amino acid mixture (glycine, alanine , valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine acid, aspartic acid, glutamic acid, lysine, arginine and histidine), 25 mM phosphocreatine, 1.7 mM dithiothreitol, 0.27 mg / mL phosphocreatine kinase, 0.03mg / mL T7 RNA polymerase, 2% polyethylene glycol, 50% volume of yeast cell extract, 15 ng / µL enhanced green fluorescent protein DNA (the DNA is prepared by traditional PCR method, and...

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Abstract

The invention discloses an optimized in vitro cell-free protein synthesis system. The system comprises a cell extract, a carbohydrate material, a phosphate compound, a buffering agent and a DNA molecular template for encoding an exogenous protein, wherein the cell extract is a yeast cell extract inserted into a T7 RNA polymerase gene; the carbohydrate material is a mixture of glucose and maltodextrin; the buffering agent is a trihydroxymethyl aminomethane buffering agent; and the DNA molecule template is prepared by a nucleic acid isothermal amplification method, and a sequence as shown in SEQID NO.1 is inserted into the upstream of the coding sequence of the exogenous protein in the DNA molecular template. By optimizing, the cost of in vitro protein synthesis is reduced and the yield ofthe target protein is increased.

Description

technical field [0001] The invention belongs to the technical field of protein synthesis, and in particular relates to a cell-free protein synthesis system for in vitro protein synthesis. Background technique [0002] Proteins are important molecules in cells and are involved in almost all functions of cells. Proteins differ in sequence and structure, determining their differing functions (1). In cells, proteins can act as enzymes to catalyze various biochemical reactions, act as signaling molecules to coordinate various activities of organisms, support biological forms, store energy, transport molecules, and enable organisms to move (2). In the field of biomedicine, protein antibodies, as targeted drugs, are an important means of treating diseases such as cancer (1,2). [0003] The traditional protein expression system refers to a molecular biology technique that expresses foreign genes through model organisms such as bacteria, fungi, plant cells or animal cells (3). Wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00
CPCC12P21/00C12N15/63C12N15/85C12N5/10
Inventor 郭敏徐开杨宁章小铃周子鉴于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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