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In-vitro cell-free protein synthesis system and application thereof

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

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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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  • In-vitro cell-free protein synthesis system and application thereof
  • In-vitro cell-free protein synthesis system and application thereof
  • In-vitro cell-free protein synthesis system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 In vitro cell-free protein synthesis system containing glucose + maltodextrin

[0078] 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, and finally 50% by volume of yeast cell extract.

[0079]In vitro protein syn...

Embodiment 2

[0089] Example 2 In vitro cell-free protein synthesis system containing maltodextrin

[0090] 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, 0.1 mM amino acid mix, 1.7 mM dithiothreitol, 0-500 mM maltodextrin, 20 mM tripotassium phosphate, 0.002 mg / mL alpha amylase, 0.03 mg / mL T7 RNA polymerase, 2% polyethylene glycol Alcohol, and finally add 50% volume of yeast cell extract.

[0091] In vitro protein synthesis reaction: add 15 ng / µL enhanced green fluorescent protein DNA to the above system, mix well and place it in an environment of 20-30 ℃ for reaction.

[0092] Fluorescent protein activity assay: Immediately after the reaction, place in Envision 2120 multi-functional microplate reader (Perkin Elmer) to detect the intensity of fluorescent signal, and use Relative Fluorescence Unit (RFU) as t...

Embodiment 3

[0094] Example 3 In vitro cell-free protein synthesis system containing lactose

[0095] 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, 0.1 mM amino acid mixture, 1.7mM dithiothreitol, 0-200 mM lactose, 20mM tripotassium phosphate, 0.03 mg / mL T7 RNA polymerase, 2% polyethylene glycol, and finally add 50% volume of yeast cells Extract.

[0096] In vitro protein synthesis reaction: add 15 ng / µL enhanced green fluorescent protein DNA to the above system, mix well and place it in an environment of 20-30 ℃ for reaction.

[0097] Fluorescent protein activity assay: Immediately after the reaction, place in Envision 2120 multi-functional microplate reader (Perkin Elmer) to detect the intensity of fluorescent signal, and use Relative Fluorescence Unit (RFU) as the activity unit.

[0098] Figure 5 It ...

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Abstract

The present invention discloses an in-vitro cell-free protein synthesis system. The system comprises cell extract, carbohydrate and a phosphate compound, wherein the carbohydrate is maltodextrin, lactose, or a combination of the maltodextrin and glucose, or a combination of the lactose and the glucose, or a combination of the maltodextrin, the lactose and the glucose. ATP is provided for an in-vitro reaction by low-cost substances such as the glucose, the maltodextrin and the lactose instead of energy sources such as phosphoenolpyruvic acid, phosphocreatine and acetyl phosphate, so that whilethe cost is reduced, a mode of providing energy by slow release prolongs the reaction time and increases the yield of target protein.

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 vary in sequence and structure, determining their differences in function (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). W...

Claims

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Application Information

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IPC IPC(8): C12P21/00
CPCC12P21/00
Inventor 郭敏徐开赵玉莲杨宁于雪
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