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Method of using phospholipase C to extracellularly express intracellular protein

A phospholipase and protein technology, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and the use of carriers to introduce foreign genetic materials, etc., can solve the problems of high cost and cumbersome extraction process, and achieve the improvement of cell membrane permeability and wide application value effect

Pending Publication Date: 2017-05-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These proteins are usually only expressed in cells and need to use physical or chemical methods to break the cells to obtain the target protein. The subsequent extraction process is not only cumbersome but also expensive. Therefore, it is necessary to find an effective way to simplify the downstream extraction steps and reduce the cost of product purification. become the researcher's goal

Method used

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  • Method of using phospholipase C to extracellularly express intracellular protein
  • Method of using phospholipase C to extracellularly express intracellular protein
  • Method of using phospholipase C to extracellularly express intracellular protein

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1: Co-expression of L. monocytogenes phospholipase C and sucrose phosphorylase

[0046] 1. Construction of recombinant bacteria: respectively with the expression plasmid pET-24a(+)-lstplc containing the L.monocytogenes phospholipase C gene (amino acid sequence shown in SEQID NO.1) and the sucrose phosphorylase gene preserved in the laboratory The expression plasmid pET-24a(+)-sp was used as a template, and PCR amplification obtained phospholipase C gene lstplc and sucrose phosphorylase gene sp containing Nco I and Hind III restriction sites, and then according to figure 1 (a) and (b) Plasmid construction process Insert L.monocytogenes phospholipase C and sucrose phosphorylase genes into the multiple cloning site MCS1 or MCS2 of the pETDuet-1 (purchased from Novagen) expression vector, respectively, and transform the ligated product The cloning host E.coli JM109 was coated with LB-Amp solid medium, cultivated in a 37°C incubator for 8 hours, picked a single colon...

Embodiment 2

[0049] Example 2: Co-expression of C. perfringens phospholipase C and sucrose phosphorylase

[0050] 1. Construction of recombinant bacteria: respectively with the expression plasmid pET-24a(+)-cpplc containing the C. perfringens phospholipase C gene (amino acid sequence shown in SEQ ID NO.2) and the sucrose phosphorylase gene preserved in the laboratory The expression plasmid pET-24a(+)-sp was used as a template, and PCR amplification obtained phospholipase C gene cpplc and sucrose phosphorylase gene sp containing Nco I and Hind III restriction sites, and then referred to figure 1 (a) and (b) Plasmid construction process Insert the C. perfringens phospholipase C and sucrose phosphorylase genes into the multiple cloning site MCS1 or MCS2 of the pETDuet-1 expression vector, and transform the ligated product into the cloning host E.coli JM109 Coat LB-Amp solid medium, culture in 37°C incubator for 8 hours, pick a single colony, and after 37°C culture in LB-Amp liquid medium for ...

Embodiment 3

[0052] Example 3: Co-expression of B. cereus phospholipase C and sucrose phosphorylase

[0053] 1. Construction of recombinant bacteria: respectively with expression plasmid pET-24a(+)-bcplc containing B. cereus phospholipase C gene (amino acid sequence shown in SEQ ID NO.3) and laboratory preservation containing sucrose phosphorylase The expression plasmid pET-24a(+)-sp of the gene was used as a template, and PCR amplification obtained phospholipase C gene bcplc and sucrose phosphorylase gene sp containing Nco I and Hind III restriction sites, and then referred to figure 1 (a) and (b) Plasmid construction process Insert the B. cereus phospholipase C and sucrose phosphorylase genes into the multiple cloning site MCS1 or MCS2 of the pETDuet-1 expression vector, and transform the ligated product into the cloning host E.coli JM109 Coat LB-Amp solid medium, culture in 37°C incubator for 8 hours, pick a single colony, and after 37°C culture in LB-Amp liquid medium for 8 hours, coll...

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Abstract

The invention discloses a method of using phospholipase C to extracellularly express intracellular protein and belongs to the technical field of bioengineering. Through co-expression phospholipase C, intracellular positioning protein is released to the outsides of cells by means of nonspecific leaking, and high-performance phospholipase C and co-expression recombinant bacteria high in target protein extracellular enzyme activity are obtained by screening. Enzyme activity of recombinant bacteria extracellular sucrose phosporylase co-expressing sucrose phosphorylase and phospholipase C is 1445.1U mL-1, and enzyme activity of recombinant bacteria extracellular trehalase co-expressing trehalase and phospholipase C is 322.3U mL-1. The method can realize extracellular expression of intracellular positioning protein, improve production efficiency and simplify post-extraction process, has wide application value in food, cosmetics and pharmaceuticals industry and has great demand in market at home and abroad.

Description

technical field [0001] The invention relates to a method for expressing intracellular protein extracellularly by using phospholipase C, which belongs to the technical field of bioengineering. Background technique [0002] Escherichia coli (Escherichia coli) expression system, as one of the most widely used and most thoroughly studied expression systems, has significant advantages in terms of length of culture period and ease of experimental operation. Since Escherichia coli has a two-layer cell membrane structure, the expression products of foreign genes in the Escherichia coli expression system may be located in different positions in the spatial structure of Escherichia coli, and there are usually three positioning methods: intracellular, extracellular and periplasmic spatial positioning. [0003] It has been found that when a naturally localized protein is recombinantly expressed in the E. coli expression system, even if a signal peptide is added to the target protein, it...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/10C12N9/24C12R1/19
CPCC12N9/16C12N9/1051C12N9/2402C12N15/70C12Y204/01007C12Y301/04003C12Y302/01028
Inventor 吴敬宿玲恰于林港
Owner JIANGNAN UNIV
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