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Optimization of Expression Conditions of Hybrid Antimicrobial Peptide LPCB in Pichia pastoris(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

2012 01
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Optimization of Expression Conditions of Hybrid Antimicrobial Peptide LPCB in Pichia pastoris
LI Zengting XIE Li FENG Shunli YOU Fangfang HE Guihua LIN Qiang CAO Yi QIAO Dairong
(1Microbiology and Metabolic Engineering Key Laboratory of Sichuan Province, College of Life Sciences, Sichuan University, Chengdu 610065, China)
(2Luohe Entry-exit Inspection and Quarantine Bureau, Luohe 462000, Henan, China)
hybrid antimicrobial peptide Lfcin-Pro-CecropinB Pichia pastoris expression condition optimization
Q936 : Q78

To construct recombinant yeast strains which can produce hybrid antimicrobial peptides with natural activity and determine the optimum conditions for expression, the SacⅠ-linearized recombinant expression vectors pPICZα-A-CecropinB (1~10)-Magainin2 (1~12) (pPICZα-A-CBMA) and pPICZα-A-Lfcin-Pro-CecropinB (pPICZα-A-LPCB) were transformed into Pichia pastoris X-33 by electroporation, respectively. The positive clone strains were screened with Zeocin and identified by PCR. The protein expression was induced by methanol and the antibacterial activity was tested through antibacterial experiments. Furthermore, the expression conditions of hybrid peptide LPCB were optimized and the stability of plasmid was tested by culture. The results showed that the genetic engineering strains P. pastoris X-33/pPICZα-A-CBMA and P. pastoris X-33/pPICZα-A-LPCB were successfully constructed. The expression product of the former exhibited little antimicrobial activity, while the latter exhibited relatively stronger activity. And the optimum expression conditions of LPCB were as follows: Temperature 25 ?C, pH neutral, adding 0.5% (V/V, φ) methanol to culture every 24 h, induced expression time 72 h. P. pastoris X-33/pPICZα-A-LPCB was cultured for 100 generations and no plasmid was lost. Fig 4, Ref 20


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Last Update: 2012-02-29