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KMID : 0613820020120040496
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Journal of Life Science
2002 Volume.12 No. 4 p.496 ~ p.503
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Development of a Recombinant Strain of Pichia pastoris with Antibacterial Activity
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Kang Dae-Ock
Lee Joon-Won
Hoe Gun-Young
Ahn Jong-Seog
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Abstract
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To develop a yeast strain of Pichia pastoris producing an antibacterial peptide, we have attempted the expression and secretion of an insect defensin. The nucleotide sequences corresponding to mature defensin were chemically synthesized by 6 oligomers, assembled in vitro and the synthesized gene was identified by nucleotide sequencing. The prepro sequence of yeast mating factor α1 and the defensin gene were recombined into a Pichia expression vector, pPIC9K. The resulting plasmid, pPIDE, was transformed into P. pastoris GS115 and transformants selected on histidine-deficient minimal plates were tested for antibacterial activity against Micrococcus luteus. Four strains with different antibacterial activity were selected for further analysis. Southern hybridization and RT-PCR verified the defensin gene was maintained and transcribed in a host. Four strains were cultivated in YPD broth for 96 hours to compare cell growth and antibacterial activity. They showed no difference in cell growth, however, each strain showed different antibacterial activity pattern with culture time. The maximal activity was about 550 AU/㎖.
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KEYWORD
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Defensin, Pichia pastoris, antibacterial activity, secretion, mating factorα1
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