Micromolecular saccharide-containing system for in-vitro high-efficiency protein synthesis

A small molecule sugar and protein technology, applied in the field of efficient protein synthesis system, to achieve the effect of simple and fast method, increase yield, and meet research requirements

Inactive Publication Date: 2018-09-07
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of developing protein drugs, it is necessary to prepare a large amount of high-quality proteins for protein s

Method used

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  • Micromolecular saccharide-containing system for in-vitro high-efficiency protein synthesis
  • Micromolecular saccharide-containing system for in-vitro high-efficiency protein synthesis
  • Micromolecular saccharide-containing system for in-vitro high-efficiency protein synthesis

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0031] Example 7

[0032] Preparation of cell disruption products:

[0033] The cultured Escherichia coli (Escherichia coli BL21(DE3) CICC 23796, purchased from CICC) was collected, suspended, centrifuged, mechanically broken, and stored.

[0034] In addition to mechanical methods, cell disruption methods can also be repeated freezing and thawing methods, ultrasonic treatment methods, enzymatic methods, alkaline lysis methods or chemical permeation methods.

[0035] The crushing process uses an ultrasonic cell pulverizer with a power of 20W, ultrasonic for 5s per cycle, 10s stop, and a total time of 1h (10-15mL is taken each time for crushing).

[0036] Examples 8-10 are composition 1, see Table 3.

[0037] table 3

[0038]

[0039] Examples 11-13 are amino acid aqueous solutions, see Table 4

[0040] Table 4

[0041]

[0042] Examples 14-16 are reaction buffers, see Table 5

[0043] table 5

[0044]

[0045] Examples 17-19 are energy supplement liquids, see Table 6

[0046]

Example Embodiment

[0047] Examples 20-22 are composition 2 (each component is volume ratio), see Table 7

[0048]

Example Embodiment

[0049] Example 23

[0050] A system for efficiently synthesizing proteins in vitro containing small molecular sugars, with a volume ratio of 20:25:1: (1.5, 3, 4.5, 6, 7.5), and the ratio includes composition 1 (prepared in Example 8) and composition 2 (Prepared in Example 20), gene (pRset-eGFP (purchased from: Promega Corporation)) and 1.334g / ml sorbose aqueous solution;

[0051] The preparation process is: mix composition 1, composition 2, gene and 5 volumes of 1.334g / ml sorbose aqueous solution, put them in a 96-well plate, react at 30°C for 12 hours, and perform detection to obtain protein expression The amount varies with the concentration of sorbose as follows figure 1 Shown.

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Abstract

The invention discloses a micromolecular saccharide-containing system for in-vitro high-efficiency protein synthesis. The system comprises a composition 1, a composition 2, a gene and a micromolecularsaccharide water solution, wherein the composition 1 comprises a cell crushing product, a buffer solution 1 and a buffer solution 2; the composition 2 comprises an amino acid water solution, a reaction buffer solution, an energy supplementing solution and a ribonucleic acid polymerase water solution with concentration of 5 to 150mug/ml. The system has the advantages that the in-vitro protein output by the cell-free protein synthesis system is increased, the method is simple, convenient and rapid, and the current study requirements of protein structure and functional proteomics can be met.

Description

technical field [0001] The invention relates to a system for efficiently synthesizing protein in vitro by adding small molecular sugars, belonging to the field of biosynthesis. Background technique [0002] The cell-free protein synthesis system is a means of rapid and efficient protein synthesis in vitro. It completes the synthesis of target proteins by adding substrates and energy required for transcription and translation to cell extracts. Compared with in vivo expression, the cell-free protein expression system has obvious advantages: a large amount of toxic protein can be expressed without being limited by cell physiology; the product is not degraded by intracellular proteases; the reaction conditions can be controlled, etc. In recent years, various proteins that are difficult to express by conventional intracellular means have been successfully expressed in cell-free cells in vitro (Goerke AR, Swartz JR. Development of cell-free protein synthesis platforms for disulfid...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/70
CPCC12N15/63C12N15/70
Inventor 仰大勇白丽慧郭小翠
Owner TIANJIN UNIV
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