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Signal peptide capable of effectively improving protein secretion efficiency and application of signal peptide

A protein secretion and signal peptide technology, applied in the field of genetic engineering, can solve problems such as difficulty in secretion and expression of heterologous proteins, and achieve the effects of increased secretion, secretion and expression, and improved enzyme activity

Inactive Publication Date: 2018-04-20
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the Bacillus subtilis expression system, proteins derived from Gram-positive bacteria can generally be effectively expressed and secreted using their own promoters and signal peptides, and the expressed products are also resistant to the proteases of the host bacteria, but heterologous proteins The secretory expression of Bacillus is more difficult, and it usually needs to be fused with the Bacillus extracellular protein mover and signal sequence to make the secretory expression effective

Method used

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  • Signal peptide capable of effectively improving protein secretion efficiency and application of signal peptide
  • Signal peptide capable of effectively improving protein secretion efficiency and application of signal peptide
  • Signal peptide capable of effectively improving protein secretion efficiency and application of signal peptide

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1 Screening of Signal Peptides Efficiently Secreted and Expressed by β-galactosidase

[0029] With reference to the method of (Christian Degering et.Al, 2010), by constructing the expression vector that various signal peptides combine with β-galactosidase coding gene (bgaB) from Bacillus subtilis homologous protein signal peptide library, combine A high-throughput screening method to obtain signal peptides that improve the secretion effect of β-galactosidase to varying degrees from clones, specifically including the following steps:

[0030] (1) Construction of pBEp43-biobrick-bgaB vector: The PCR fragment of biobrick (biological building block) obtained by annealing and extending two artificially synthesized fragments of SEQ ID NO.3 and SEQ ID NO.4 has restriction sites KpnI and XhoI, and used Restriction endonuclease digested and purified 100bp PCR product (such as figure 1 ) was inserted into the plasmid pBE-P43-bgaB (according to the patent literature: Pan ...

Embodiment 2

[0035] The detection of embodiment 2 signal peptide expression levels

[0036] (1) Construction of MTG (transglutaminase) extracellular expression plasmid: the plasmid pBEp43-proMTG (according to the patent literature: Pan Li et al. A recombinant Bacillus subtilis and its method for producing transglutaminase. CN201210052578 .2 [P]. 2012. Construction) as an expression plasmid, using the pBEp43-SPyoqM-bgaB plasmid obtained in the above-mentioned Example 1 as a template, primers F-SPyoqM (5'-GAGAGGAATGTCGACATGAA ATTAAGAAAAGTATTGACTG-3'), R-SPyoqM (5 '-CGTTGTCCATCTCGAGAGCGAATGCAGGAGAAGCAGAAAC-3') to amplify about 100bp SPyoqM fragment (see image 3 ). The signal peptide (SPyoqM fragment) was connected with the linearized plasmid (pBEp43-proMTG) by the In-fusion method (see the HiFi DNA Assembly Master Mix of NEBuilder for the specific operation method), and the MTG expression extracellular plasmid pBEp43-SPyoqM-proMTG was constructed (see Figure 4 ). First transform into Esc...

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Abstract

The invention discloses a signal peptide capable of effectively improving protein secretion efficiency and an application of the signal peptide. The amino acid sequence of the signal peptide is shownin SEQ ID NO.1. The nucleotide sequence for encoding the signal peptide is selected from any of the following sequences: (a) a nucleotide sequence shown in SEQ ID NO.2 or a complementary sequence of the nucleotide sequence; and (b) a nucleotide sequence which is obtained through substitution, deletion or addition of one or more nucleotides of the nucleotide sequence shown in SEQ ID NO.2, has a same function of being used a signal peptide with the nucleotide sequence shown in SEQ ID NO.2, or a complementary sequence of the obtained nucleotide sequence. According to the invention, an expressionvector with the combination of the signal peptide and beta-galactosidase encoding gene / transglutaminase zymogen encoding gene is constructed from a vacillus subtilis homologous protein signal peptidelibrary, the signal peptide capable of increasing the secretion of beta-galactosidase can be screened out, and the secretion expression of the transglutaminase zymogen is realized.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a signal peptide capable of effectively improving protein secretion efficiency and its application. Background technique [0002] Bacillus subtilis (Bacillus subtilis) is a kind of Gram-positive aerobic bacilli that can endogenously resist spores. The composition of the cell wall is simple, does not contain endotoxin, and has only a single outer membrane. Therefore, when the secreted protein passes through the cell membrane , and after being processed and folded in the intercellular matrix, it is directly released into the culture medium. Because it does not require high culture conditions and can maintain a high bacterial concentration in large-scale production, it is widely used in the production of industrial enzymes. And its genetics and physiology have been deeply studied, and people's understanding of its genetic background and physiological characteristics is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/32C12N15/31C12N15/75C12N1/21C12R1/125
Inventor 潘力刘欣叶燕锐王斌
Owner SOUTH CHINA UNIV OF TECH
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