Functional expression and molecular characterization of Culex quinquefasciatus salivary α-glucosidase (MalI)

Protein Expression and Purification
Rungarun SuthangkornkulDumrongkiet Arthan

Abstract

Salivary α-glucosidases (MalI) have been much less characterized when compared with midgut α-glucosidases, which have been studied in depth. Few studies have been reported on the partial characterization of MalI, but no clear function has been ascribed. The aim of this study is to purify and characterize the recombinant Culex quinquefasciatus (CQ) α-glucosidase expressed in Pichia pastoris. The cDNA encoding mature Cx. quinquefasciatus α-glucosidase gene with polyhistidine tag (rCQMalIHis) was successfully cloned into the expression vector, pPICZαB, designated as pPICZαB/CQMalIHis. The activity of recombinant rCQMalIHis expressed in P. pastoris could be detected at 3.75U/ml, under optimal culture conditions. The purified rCQMalIHis showed a single band of molecular weight of approximately 92kDa on SDS-PAGE. After Endoglycosidase H digestion, a single band at 69kDa was found on SDS-PAGE analysis, suggesting that rCQMalIHis is a glycoprotein. Additionally, tryptic digestion and LC-MALDI MS/MS analysis suggested that the 69kDa band corresponds to the Cx. quinquefasciatus α-glucosidase. Thus, rCQMalIHis is a glycoprotein. The rCQMalIHis exhibited optimum pH and temperature at 5.5 and 35°C, respectively. The catalytic efficiency (kc...Continue Reading

References

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