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Plectasin in prokaryotic cells and lytic activity

Author: XieMengYi
Tutor: XiaXinJie；WuDe
School: Sichuan Agricultural University
Course: Comparison of animal and human nutrition
Keywords: Plectasin Antimicrobial peptides Gene Expression Fusion protein Antibacterial activity
CLC: Q93
Type: Master's thesis
Year: 2011
Downloads: 29
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Abstract


Plectasin from saprophytic ascomycetes (Pseudoplectania nigrella) secreted protein first isolated from fungal defensin. Plectasin mature functional fragment is only 40 amino acids with a molecular weight of approximately 4.4KDa, its spatial structure of defensin typical CSap motifs. Plectasin against Gram-positive bacteria, especially Streptococcus pneumoniae has a strong bactericidal effect, and because of its unique bactericidal mechanism so Plectasin non-cytotoxic and hemolytic and other hazards, so it has a lot of drugs with potential and future large-scale production. This paper studies Plectasin expressed in E. coli and to explore ways of metal chelate affinity chromatography Plectasin best purification conditions. Plectasin NCBI GenBank under the mature peptide gene sequence, mature peptide Plectasin synthetic DNA fragment and stored in plasmid pPIC-9K. Artificial gene sequence according to its design a pair of specific primers were synthesized in order to save on pPIC-9K plasmid Plectasin gene fragment as a template by PCR amplification of the target gene was cloned and then forward into the pET-32a () expression vector construct pET32a ()-Plectasin expression plasmid. Verified by sequencing analysis of the recombinant plasmid gene sequences Plectasin correctness, will pET32a ()-Plectasin recombinant plasmids were transformed into E. coli Rosetta (DE3) expression strains, as with IPTG inducer fusion genes were induced for expression. SDS-PAGE protein electrophoresis showed that the transformed strain expressing a fusion protein of approximately 25 KDa fragments, with the expected fusion protein Trx-Plectasin match the size, the expression yield about 50% of the total protein. Broken cells, separation and extraction of the fusion protein, using Ni-IMAC purified by metal chelate affinity chromatography fusion protein detected by SDS-PAGE purity seen after a size of about 25 KDa band eluted, with the expected size of the fusion protein in line . With enterokinase cleavage of the fusion tag and release Plectasin mature peptides and recombinant Plectasin detected Staphylococcus aureus significantly inhibited. In conclusion, this study successfully achieved Plectasin genes in prokaryotic expression system of efficient expression, but also worked out to improve one metal chelate affinity chromatography purification of disulfide-rich peptides purification efficiency conditions for the rich disulfide-containing antimicrobial peptide gene engineering production of reference.

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CLC: > Biological Sciences > Microbiology
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