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Method for improving expression efficiency of protein in clostridium

A protein expression, clostridial technology, applied in the field of genetic engineering, can solve the problem of lack of fusion auxiliary protein expression efficiency and other problems, and achieve the effect of improving application value and improving expression efficiency

Active Publication Date: 2020-11-10
NANJING UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in Clostridium, whether the expression of a single protein or multiple proteins was expressed in previous studies, the gene of the protein itself was simply expressed alone, and there was no report on the fusion of auxiliary proteins to improve the expression efficiency

Method used

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  • Method for improving expression efficiency of protein in clostridium
  • Method for improving expression efficiency of protein in clostridium
  • Method for improving expression efficiency of protein in clostridium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Identification of non-canonical secreted proteins in biofilms

[0047] (1) Strain activation: Take 200 μL of C. acetobutylicum B3 (Clostridium acetobutylicum b3) glycerol bacteria solution stored at -80°C, spread it evenly on the P2 solid plate medium in a sterile ultra-clean workbench, and place it at 37 After 36 hours of static culture in a constant temperature anaerobic environment at ℃, transfer to fresh P2 solid plate medium, and static culture in a constant temperature anaerobic environment at 37 ℃ for 12 hours.

[0048] (2) Seed culture: Take an appropriate amount of the above-mentioned activated bacteria sludge, transfer it to 50mL P2 liquid seed medium in a sterile ultra-clean workbench, and place it in a constant temperature anaerobic environment at 37°C for static culture for 12h (OD 600 =2.2) is the seed solution, ready to use.

[0049] (3) Fermentation culture: The medium used in the fermentation experiment of C. acetobutylicum B3 is P2 fermenta...

Embodiment 2

[0055] Example 2: Identification of non-classical secreted proteins in biofilms by protein two-dimensional electrophoresis

[0056] Using the extracellular proteins in the biofilm obtained in step (4) of Example 1, the extracellular proteins in the biofilm were analyzed and identified by protein two-dimensional electrophoresis (2DE) and mass spectrometry. Non-canonical secreted proteins identified see figure 1 .

Embodiment 3

[0057] Example 3: Identification of non-classical secreted proteins in Clostridium acetobutylicum culture supernatant

[0058] Get the fermentation broth of Clostridium in Example 1, centrifuge and collect the supernatant sample, carry out signal peptide analysis after mass spectrometry analysis: Utilize SignalP5.0 (http: / / www.cbs.dtu.dk / services / SignalP / ) online analysis system to analyze whether the extracellular protein contains a classic secretion signal peptide sequence. Table 4 shows the non-classical secreted proteins in the culture supernatant of Clostridium acetobutylicum, and a certain comparison of their protein abundance (emPAI).

[0059] Table 4. Nonclassical Secreted Proteins of Clostridial Culture Supernatants

[0060]

[0061]

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Abstract

The invention discloses a method for improving the expression efficiency of protein in clostridium. Target protein is fused with non-classical secretory protein; and protein expression is carried outin clostridium. Compared with the prior art, the invention provides a brand-new method for improving the protein expression efficiency of recombinant protein in clostridium, and lays a technical foundation for optimizing the preparation and application of the recombinant protein in clostridium.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for improving protein expression efficiency in Clostridium. Background technique [0002] Clostridium acetobutylicum is a Gram-positive bacterium. As a traditional strain for the fermentation and production of ABE (acetone-butanol-ethanol), it is currently widely used in the research and development of bioenergy production processes and metabolic engineering. The technique of expressing the target protein by means of genetic engineering in this strain is very mature. In model strains such as Escherichia coli, researchers can improve the solubility, stability or expression level of the target protein by fusing some well-known protein tags, molecular chaperones or other auxiliary proteins, thereby improving the expression efficiency of the target protein. However, in Clostridium, whether the expression of a single protein or multiple proteins was e...

Claims

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Application Information

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IPC IPC(8): C12N15/67C12N15/74C07K19/00C12R1/145
CPCC12N15/74C07K2319/036C07K14/43595Y02E50/10
Inventor 应汉杰柳东曹幸园王振宇葛士凯陈勇牛欢青刘庆国
Owner NANJING UNIV OF TECH
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