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| Constitutive Expression of α-galactosidase Mel1 Gene in Pichia pastoris |
| ZHANGXue-wen;TANGXiang-shan;ZHANGJin-chen;ZHANGHuai-yun |
| College of Bio-science and Technology, Hunan Agricultural University, Changsha 410128, China; |
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Abstract Mel1 gene was amplified from yeast(Saccharomyces cerevisiae ) AH109 with PCR, and cloned into integrated vector pGAPZαA, constitutive and secreted expression α-galactosidase plasmid pGAPZα-Mel1 was constructed. The linearized recombinant plasmid pGAPZ α-Mel1 was transformed into Pichia pastoris KM71 by electropotation, and the blue positive colonies were screened out on YPDS plate with X-α-gal and 100 mg/mL zeocin. There was a special 53 kD band by SDS-PAGE from supernatant of yeast culture and a color band was observed by soak the native PAGE gel in X-α-gal to illustrate the α-galactosidase activity; The α-galactosidase enzyme activity was 12 U/mL after fermenting pGAPZα-Mel1 /KM71 for 6 days.
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Received: 06 April 2005
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Corresponding Authors:
ZHANG Xue- wen
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