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Peptide SMAP-29 gene design, synthesis, expression, purification and detection of antibacterial activity

Author: HanFuLang
Tutor: XiaoJianHua
School: Nanhua University
Course: Pathogen Biology
Keywords: Antimicrobial peptides SMAP-29 Gene Design Prokaryotic expression
CLC: R346
Type: Master's thesis
Year: 2006
Downloads: 177
Quote: 1
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Abstract


Objective: To design and synthesis of new antimicrobial peptide SMAP-29 gene , prokaryotic expression vector pGEX-4T-1/SMAP-29, by inducing expression and purification , to obtain a large number of peptides with antibacterial activity SMAP-29. Methods: According to SMAP-29 protein amino acid sequence , selection of E. coli preferred codons , taking into account the free energy of mRNA secondary structure to design new genes, gene using geneSOEing (gene splicing by overlap extension, geneSOEing) synthesis , synthetic genes cloned into pGEX-4T-1 prokaryotic expression vector by restriction analysis , PCR and sequencing screened positive recombinant plasmid ; recombinant plasmid was transformed into E. coli BL21 (DE3) induced by IPTG conducted by GST Sepharose FF chromatography column purification , while for thrombin cleavage , and then purified by HPLC to obtain 95% purity of the target protein . Detection of antimicrobial peptide SMAP-29 activity , using broth dilution method against Staphylococcus its ( clinical isolates ) , Enterococcus faecalis resistant strains ( resistant to fluoroquinolones ) , Enterococcus faecalis standard strain (ATCC29212) and large intestine coli (JM 109) of the MIC and MBC values. Results: geneSOEing synthesis of the new gene is 109bp ( including restriction sites ) , cloned into pGEX-4T-1 prokaryotic expression vector by restriction analysis , PCR and sequencing , the sequence is designed sequences were identical . SDS-PAGE electrophoresis showed pGEX-4T-1/SMAP-29 expressed in BL21 size of approximately 29KD GST-SMAP-29 fusion protein by thrombin cleavage and purification of the obtained size of about 3.2KDa SMAP-29 polypeptide of interest . Broth microdilution assay showed that SMAP-29 has strong antibacterial activity.

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