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
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[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. Extract the genome of Geobacillus stearothermophilus str.NO.2 (the strain number is ATCC 7953). For the extraction method, please refer to Takara Genome Extraction Kit.
[0032] 2. Primer design and PCR to obtain the cyclodextrin glucosyltransferase gene.
[0033] The primer design was designed according to the Geobacillus stearothermophilus CGTase gene sequence (GenBank accession number X59043.1) in the NCBI database, and the upstream and downstream primers containing BamHI and XhoI sites (synthesized by Shanghai Shenggong 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] (The underlined...
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[0039] Example 2 Construction and induced expression of Escherichia coli engineering bacteria producing cyclodextrin glucosyltransferase
[0040] 1: Construction and transformation of recombinant plasmids.
[0041] Use restriction enzymes BamHI and XhoI to cut the PCR product of the cyclodextrin glucosyltransferase gene obtained in the previous step. The restriction enzyme digestion system is: PCR product DNA 5μL, BamHI 0.5μL, XhoI 0.5μL, 10×H buffer 2μL , Add double distilled water to 20μL; the digestion system for recovery is: plasmid DNA 16μL, Nco I 1μL, EcoR I 1μL, 10×Hbuffer 2μL. Perform 1% agarose gel electrophoresis to detect the digested product or recover the target fragment. At the same time, the plasmid pET-20b(+) is subjected to the same double digestion treatment, and then the digested product is recovered by the gel.
[0042] To connect the insert and plasmid, use a ligation kit. The vector and the insert are mixed at a molecular ratio of 1:1 to 1:10, and an equal am...
Example Embodiment
[0044] Example 3 Preparation of signal peptide mutants containing different DB sequences
[0045] The 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 are designed to be thirteen degenerate bases using the degeneracy of the codon, and the pET-20b(+)-CGT plasmid is used as a template, On the basis of the nucleotides shown in the sequence SEQ ID NO.3, the F2primer with the sequence shown in SEQ ID NO.7 and the R2primer with the sequence shown in SEQ ID NO.8 are primers, and PCR is performed to mutate into: TGTATGTATTACA, get the recombinant plasmid pET-20b(+)-DB1-CGT, use the codon degeneracy, and also get the recombinant plasmid pET-20b(+)-DB2-CGT, the DB2 signal peptide nucleotide sequence is as SEQ ID Shown in NO.9. (The structure diagram of recombinant plasmid pET-20b(+)-DB1-CGT is as follows figure 1 As shown; PCR verific...
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