Gene cloning and expression of taro α-galactosidase

• Main Authors: Houng-Yi Lee, 李皇毅
• Other Authors: 簡素芳
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/20004604989173897779

碩士 === 淡江大學 === 生命科學研究所碩士班 === 98 === α-Galactosidase is capable of hydrolyzing terminal non-reducing galactosyl residues, and it is distributed in most organisms with many different biological funtions such as metabolism and transportation (source to sink transport) of photoassimilates in plants. In application, α-galactosidase can be used to hydrolyze the galactosyl group of type B antigen on the red blood cell surface, and covert the type B red blood cell into type O. Thus, the Pichia patoris expression system was used to produce large amount of taro α-galactosidase fastly to proceed the seroconversion of erythrocyte. The crude taro α-galactosidase was extracted with 83.3 Unit/kg of enzyme activity from taro tubers. The crude extracts then purified to homogeneity by using column chromatography, and the specific activity was 54.18 Unit/mg after 6019 fold purification. Base on the results of N-terminal sequencing, inner peptide determination, and multiple alignment with GH27 family α-galactosidase, degenerated primers dFP IA and dRP 2A were designed to synthesis the first known sequence of taro α-galactosidase gene. Then forward and reverse gene specific primers were designed from the known sequence to obtain the whole gene sequence, which was consisted of 1082 bases, and the molecular mass of this enzyme was 40.01 kDa, by PCR techniques. The gene was cloned into pPIC9k, and then transformed into Pichia pastoris (GS115 and SMD1168). At last, Pichia pastoris was induced and expressed recombinant taro α-galactosidase. After the recombinant gene had been induced for six days, the intracellular enzyme activities of GS115 and SMD1168 were 0.5 and 0.45 Unit/mL(culture) at most, and the extracellular enzyme activities were only 1/10 fold of the intracellular activities.