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Signal peptide for promoting protein extracellular expression

A signal peptide and protein technology, applied in the fields of genetic engineering and enzyme engineering, can solve the problems of changing catalytic properties and limited improvement

Pending Publication Date: 2020-10-30
JILIN COFCO BIOCHEM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the mutation of pullulanase itself, although it can increase its extracellular expression, it often changes its catalytic properties; while the addition of a signal peptide can increase the extracellular expression of the enzyme, but the increase is still limited

Method used

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  • Signal peptide for promoting protein extracellular expression
  • Signal peptide for promoting protein extracellular expression
  • Signal peptide for promoting protein extracellular expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Construction of Ggt signal peptide NCS synonymous mutation library

[0050]In order to promote the high-efficiency expression of pullulanase, the Bacillus subtilis transcription LytR promoter (shown in SEQ ID NO.4 for sequence information) with the highest endogenous transcription level characterized by previous laboratories was passed through a one-step cloning kit (purchased Connected to the P43NMK plasmid from Nanjing Nuoweizan Biotechnology Co., Ltd. to construct a recombinant plasmid, transfer the recombinant plasmid into E.coli JM109, and spread the bacterial solution on an LB plate containing 50 μg / mL ampicillin resistance , cultivated at 37°C until a single clone was grown, and the single clone was picked out and verified by plasmid sequencing after culture to obtain the plasmid P43NMK-LytR; using the same one-step cloning method, the pullulanase gene (nucleotide sequence such as SEQ ID NO .6) was fused to the downstream of LytR by a one-step cloning ...

Embodiment 2

[0052] Example 2: Construction of YweA signal peptide NCS synonymous mutation library

[0053] The specific embodiment is the same as in Example 1, the difference is that after obtaining P43NMK-LytR-Pul, the sfGFP fluorescent protein is connected to the C-terminus of pullulanase by a one-step cloning method, and the YweA signal peptide wild-type (nucleotide The sequence is shown in SEQ ID NO.9) was connected to the N-terminal of pullulanase to construct the recombinant plasmid P43NMK-YweA-Pul.

[0054] Using P43NMK-YweA-Pul as a template, using degenerate primers (nucleotide sequences shown in SEQ ID NO.11 and SEQ ID NO.13), PCR obtained a synonymous mutation library of the first 30 bases of the N-terminal of YweA ( Synonymous mutation recombinant plasmid), that is, the first 30 amino acid sequences remain unchanged, and the nucleotide sequence is changed.

Embodiment 3

[0055] Example 3: Construction of YolA signal peptide NCS synonymous mutation library

[0056] The specific embodiment is the same as in Example 1, the difference is that after obtaining P43NMK-LytR-Pul, a one-step cloning method is used to connect the sfGFP fluorescent protein to the C-terminus of pullulanase, and the YolA signal peptide wild-type (nucleotide The sequence is shown in SEQ ID NO.10) was connected to the N-terminal of pullulanase to construct the recombinant plasmid P43NMK-YolA-Pul.

[0057] Using P43NMK-YolA-Pul as a template, using degenerate primers (nucleotide sequences shown in SEQ ID NO.11 and SEQ ID NO.14), PCR obtained a synonymous mutation library of the first 30 bases of the N-terminal of YolA ( Synonymous mutation recombinant plasmid), that is, the first 30 amino acid sequences remain unchanged, and the nucleotide sequence is changed.

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Abstract

The invention discloses a signal peptide for promoting protein extracellular expression, and belongs to the technical field of gene engineering and enzyme engineering. According to the invention, bacillus subtilis is used as a host, and the method comprises the following steps: performing synonymous mutation on endogenous signal peptides Ggt, YweA and YolA of bacillus subtilis, selecting monoclones with high and low fluorescence values through high-throughput screening, and after sequencing identification and signal peptide mutation, inoculating variants into a shake flask for fermentation verification, so that a synonymous mutation sequence capable of remarkably improving the extracellular protein quantity is obtained, and the protein expression quantity can be increased while normal protein translation is guaranteed. The synonymous mutation sequences of the Ggt signal peptide, the YweA signal peptide and the YolA signal peptide respectively enable the extracellular enzyme activity tobe improved by 1.45 times, 0.59 times and 1.04 times compared with that of a wild type, so that a new strategy is provided for extracellular high-efficiency expression of enzymes.

Description

technical field [0001] The invention relates to a signal peptide for promoting protein extracellular expression, belonging to the technical fields of genetic engineering and enzyme engineering. Background technique [0002] Pullulanase (Pullulanase, EC 3.2.1.41) is a widely used starch debranching enzyme, which can hydrolyze starch polysaccharides such as pullulan, pullulan, α-dextrin, β-dextrin, sugar Alpha-1,6 glycosidic linkages in native and related polysaccharides. Pullulanase has been widely used in the production of ethanol fuel, cyclodextrin, resistant starch, maltotriose syrup and other products. Because of its polysaccharide-hydrolyzing properties, the expression system for pullulanase should preferably be constructed in a food-grade expression system. [0003] Bacillus subtilis has important applications in the food industry, antibody production and animal husbandry. However, the expression level of pullulanase in Bacillus subtilis is very low at present, and i...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/75C12N15/62C12N1/21C12N9/44C12R1/125
CPCC07K14/32C12N15/75C12N9/2457C12Y302/01041C07K2319/02
Inventor 佟毅刘松徐奎栋李江华陈坚
Owner JILIN COFCO BIOCHEM
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