Signal peptide mutant for improving secretion efficiency of protein and application thereof

A signal peptide and mutant technology, applied in the field of genetic engineering, can solve the problems of unable to achieve secretion expression efficiency and limited ability, and achieve the effect of efficient secretion and high-efficiency protein

Active Publication Date: 2017-08-08
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The vast majority of secreted proteins are transported out of the intracellular membrane to the periplasm through the SecB-dependent pathway. However, signal peptides commonly used in this pathway, such as PelB, OmpA, PhoA, etc., have limited ability to promote secretion and expression, and cannot achieve the ideal increase in secretion. The effect of expressive efficiency

Method used

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  • Signal peptide mutant for improving secretion efficiency of protein and application thereof
  • Signal peptide mutant for improving secretion efficiency of protein and application thereof
  • Signal peptide mutant for improving secretion efficiency of protein and application thereof

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Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1 Contains the construction of mutant pelB signal peptide vector

[0032] Using the pET-20b(+) vector as a template, using F1primer whose sequence is shown in SEQ ID NO.5 and R1primer whose sequence is shown in SEQ ID NO.6 as primers, perform PCR to mutate the three amino acids MKY at the amino terminal of the pelB signal peptide For MKH, the recombinant plasmid pET-20b(+)-pelBH was obtained, and primers with sequences such as SEQ ID NO.11 and SEQ ID NO.12 were used to mutate the third amino acid tyrosine on the pelB signal peptide into a sperm amino acid to obtain the recombinant plasmid pET-20b(+)-pelBR; the third amino acid tyrosine on the pelB signal peptide is mutated into lysine with primers whose sequences are shown in SEQ ID NO.13 and SEQ ID NO.14 respectively amino acid to obtain the recombinant plasmid pET-20b(+)-pelBK. The mutated pelBR and pelBK signal peptide nucleotide sequences are respectively shown in SEQ ID NO.9 and SEQ ID NO.10), and then ...

Embodiment 2

[0035] Example 2 Acquisition of Cyclodextrin Glucosyltransferase Gene

[0036]The target protein used in the present invention is cyclodextrin glucosyltransferase, which is obtained by PCR.

[0037] 1. Genome extraction of Geobacillus stearothermophilus str.NO.2 (the strain number is ATCC 7953). For the extraction method, see Takara Genome Extraction Kit.

[0038] 2. Primer design and PCR to obtain cyclodextrin glucosyltransferase gene.

[0039] The primer design was designed according to the Geobacillus stearothermophilus CGTase gene sequence in the NCBI database (GenBank accession number is X59043.1), and the upstream and downstream primers containing BamHI and XhoI sites respectively (synthesized by Shanghai Sangon Bioengineering):

[0040] Upstream primer F2primer: 5'-CGCGGATCCGCAATCTTCATCGTGTCCGACACCCAAAAG-3' (the underlined sequence is the BamHI restriction site);

[0041] Downstream primer R2primer: 5'-CCGCTCGAGTTAATTCTGCCAATCCACGATAATTTTGCCGGT-3' (the underlined sequ...

Embodiment 3

[0044] Example 3 Cyclodextrin Glucosyltransferase-producing Escherichia coli Engineering Bacteria Construction and Induced Expression

[0045] 1: Construction and transformation of recombinant plasmids.

[0046] Digest the PCR product of the cyclodextrin glucosyltransferase gene obtained in the previous step with restriction endonucleases BamHI and XhoI. The enzyme digestion system for verification is: PCR product DNA 5 μL, BamHI 0.5 μL, XhoI 0.5 μL, 10×Hbuffer 2 μL, Add double distilled water to 20 μL; the digestion system for recovery is: 16 μL of plasmid DNA, 1 μL of Nco I, 1 μL of EcoR I, 2 μL of 10×Hbuffer. Perform 1% agarose gel electrophoresis to detect the digested product or recover the target fragment. At the same time, the recombinant plasmids pET-20b(+)-pelBH, pET-20b(+)-pelBR, and pET-20b(+)-pelBK were subjected to the same double digestion treatment, and then the digested products were recovered by gel.

[0047] Ligate the insert and the plasmid using a ligatio...

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Abstract

The invention discloses a signal peptide mutant for improving secretion efficiency of protein and application thereof, and belongs to the technical field of gene engineering. The known signal peptide PelB is modified; the tyrosine at the site 3 of the PelB signal peptide is mutated into histidine, so that the activity of cyclodextrin glucanotransferase and the extracellular secretion efficiency are obviously improved. The signal peptide mutant for improving secretion efficiency of protein has the advantages that the activity of the target protein cyclodextrin glucanotransferase is improved by 1.4 times, the ability of extracellular secretion protein of recombinant bacteria is improved, and the extracellular expression of conventional protein is facilitated.

Description

technical field [0001] The invention specifically relates to a signal peptide mutant for improving protein secretion efficiency and application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Cyclodextrin glucosyltransferase (abbreviated as CGTase, EC 2.4.1.19) is an extracellular enzyme that can convert starch and related substrates to synthesize cyclodextrin through intramolecular transglycosylation reaction. Cyclodextrin glucosyltransferase has a wide range of applications and is mainly used to catalyze the production of cyclodextrin. Cyclodextrin glucosyltransferase converts starch to cyclodextrins by cyclization. In addition to catalyzing the production of cyclodextrins, cyclodextrin glucosyltransferases have other roles. With the increasing application of cyclodextrins in food, medicine, cosmetics, agriculture and other fields, CGTase has become a hot research topic today. [0003] To overcome the low CGTase production...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/11C12N15/67C12N1/21C12N9/10C12R1/19
CPCC07K14/00C12N9/1074C12N15/67C12N2800/70C12Y204/01019
Inventor 堵国成刘龙陈坚李江华邓琛
Owner JIANGNAN UNIV
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