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Updated: Jun 23, 2026
Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
[Cloning of the gene encoding alpha-glucosidase from aspergillus niger and its expression in Pichia pastoris]
Xing Tong1, Qiusong Tang, Yufei Wu
1State Key Laboratory of Food Science and Technology of China, Jiangnan University, Wuxi 214122, China. tongxing293@hotmail.com
Objective:
Alpha-glucosidase from Aspergillus niger SG136 was expressed in Pichia pastoris.
Methods:
cDNA of mature alpha-glucosidase (aglu) was amplified from the total DNA of A. niger SG136 by PCR and overlap-PCR with primers designed based on the sequence of A. niger CBS 513.88 in NCBI database. The gene was cloned into pMD18-T simple vector. The sequencing result showed that the gene encoded for a protein of 960 amino acids residues, which was 1 amino acid different from that of A. niger CBS 513.88. The expression vector pPIC9K-aglu was constructed by subcloning the gene into plasmid pPIC9K, and then transformed into P. pastoris through electroporation after linearized by Bgl II digestion. The recombinant P. pastoris KM71/pPIC9K-aglu were screened in MD and YPD/G418 plates,and identified by PCR. In shaking culture condition, methanol was added to a final concentration of 1% to induce the secretion of alpha-glucosidase.
Results:
Electrophoresis analysis of KM71/pPIC9K-aglu culture supernatant showed that there were two major protein bands corresponding to 98 kDa and 33 kDa respectively in SDS-PAGE, and there was only one band in Native PAGE; while in the control experiment of KM71/pPIC9K, there were no visible bands. Transglucosidation reaction from crude enzyme revealed that contents of isomaltooligosaccharides were up to 26.0% under the optimal conditions of pH 5 and 60 degrees C at 24 h.
Conclusion:
A. niger alpha-glucosidase was expressed in P. pastoris with transglucosidation activity.
