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PelB signal peptide mutant capable of improving protein secretion efficiency and application of pelB signal peptide mutant

A technology of protein secretion and signal peptide, applied in the field of genetic engineering, can solve the problems of limited improvement of signal peptide and limited production of cyclodextrin glucosyltransferase, etc., to achieve the effect of high-efficiency protein

Active Publication Date: 2017-08-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is very limited to increase the yield of cyclodextrin glucosyltransferase through simple heterologous expression, and it is often necessary to further enhance its heterologous expression through molecular modification
The pelB signal peptide has been used to increase the secreted expression of cyclodextrin glucosyltransferase, however, the ability of this signal peptide to increase expression is still limited

Method used

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  • PelB signal peptide mutant capable of improving protein secretion efficiency and application of pelB signal peptide mutant
  • PelB signal peptide mutant capable of improving protein secretion efficiency and application of pelB signal peptide mutant
  • PelB signal peptide mutant capable of improving protein secretion efficiency and application of pelB signal peptide mutant

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Embodiment 1

[0029] Example 1 Acquisition of Cyclodextrin Glucosyltransferase Gene

[0030] The target protein used in the present invention is cyclodextrin glucosyltransferase, which is obtained by PCR.

[0031] 1. Genome extraction of Geobacillus stearothermophilus str.NO.2 (the strain number is ATCC 7953). For the extraction method, see Takara Genome Extraction Kit.

[0032] 2. Primer design and PCR to obtain cyclodextrin glucosyltransferase gene.

[0033] 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):

[0034] Upstream primer F1primer: 5'-CGC GGATCC GCAATCTTCATCGTGTCCGACACCCAAAAG-3' (the underlined sequence is the BamHI restriction site);

[0035] Downstream primer R1primer5'-CCG CTCGAG TTAATTCTGCCAATCCACGATAATTTTGCCGGT-3'

[0036] (Th...

Embodiment 2

[0039] Example 2 Construction and induced expression of Escherichia coli engineering bacteria producing cyclodextrin glucosyltransferase

[0040] 1: Construction and transformation of recombinant plasmids.

[0041]Digest the PCR product of the cyclodextrin glucosyltransferase gene obtained in the previous step with restriction endonucleases BamHI and XhoI. The digestion system for verification is: PCR product DNA 5 μL, BamHI 0.5 μL, XhoI 0.5 μL, 10×H buffer 2 μL , add double distilled water to 20 μL; the enzyme digestion system for recovery is: plasmid DNA 16 μL, Nco I 1 μL, EcoR I 1 μL, 10×Hbuffer 2 μL. Perform 1% agarose gel electrophoresis to detect the digested product or recover the target fragment. At the same time, the plasmid pET-20b(+) was subjected to the same double digestion treatment, and then the digested product was recovered by gel.

[0042] Ligate the insert and the plasmid using a ligation kit. The vector and insert fragments were mixed at a molecular rati...

Embodiment 3

[0044] Example 3 Preparation of signal peptide mutants containing different DB sequences

[0045] The signal peptide of signal peptide pelB (shown in SEQ ID NO.3) was used as the starting sequence for mutation.

[0046] The thirteen amino acids CCTGCTGCCGACC at positions +9 to +21 downstream of the start codon on the vector were designed as thirteen degenerate bases using codon degeneracy, and the pET-20b(+)-CGT plasmid was used as a template, On the basis of the nucleotides shown in the sequence SEQ ID NO.3, the F2primer whose sequence is shown in SEQ ID NO.7 and the R2primer whose sequence is shown in SEQ ID NO.8 are used as primers, and PCR is carried out to mutate into: TGTATGTATTACA, obtain recombinant plasmid pET-20b(+)-DB1-CGT, utilize the degeneracy of codon, also obtain recombinant plasmid pET-20b(+)-DB2-CGT at the same time, DB2 signal peptide nucleotide sequence such as SEQ ID Shown in NO.9. (The structural diagram of the recombinant plasmid pET-20b(+)-DB1-CGT is ...

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Abstract

The invention discloses a pelB signal peptide mutant capable of improving the protein secretion efficiency and an application of the pelB signal peptide mutant and belongs to the technical field of gene engineering. A sequence of a pelB mutant secretive expression signal peptide is as shown in SEQ ID NO.1. The signal peptide is capable of improving the extracellular enzyme activity of objective protein cyclodextrin glycosyltransferase by 1.6 times. The extracellular protein production capability of recombinant escherichia coli transformed by using the signal peptide is strengthened, and industrialized production is facilitated.

Description

technical field [0001] The invention relates to a pelB signal peptide mutant capable of improving protein secretion efficiency and application thereof, belonging to the technical field of genetic engineering. Background technique [0002] Cyclodextrin glucosyltransferase (CGTase for short) 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. For example, cyclodextrin glucosyltransferase is used as a preservative in bread baking, which can extend the shelf life of food. In addition, cyclodextrin glucosyltransferase can be used to catalyze the transfer of one or more sugar groups to carbohydrates to improve their properties (such as increased solubility, stability, reduced cytotoxicity, bitterness, etc.). [0003] The production of cyclodextr...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/11C12N15/70C12N1/21C12N9/10C12R1/19
CPCC07K14/00C12N9/1074C12N15/70C12N2810/40C12Y204/01019
Inventor 堵国成刘龙陈坚李江华邓琛
Owner JIANGNAN UNIV
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