Functional expression and molecular characterization of Culex quinquefasciatus salivary α-glucosidase (MalI)
| Autor: | Phanthila Sirichaiyakul, Sungsit Sungvornyothin, Dumrongkiet Arthan, Rungarun Suthangkornkul, Apanchanid Thepouyporn, Jisnuson Svasti |
|---|---|
| Rok vydání: | 2014 |
| Předmět: |
DNA
Complementary Recombinant Fusion Proteins Gene Expression Pichia Salivary Glands law.invention Pichia pastoris Substrate Specificity chemistry.chemical_compound Endoglycosidase H law Complementary DNA Maltotriose Animals Histidine Cloning Molecular Glycoproteins chemistry.chemical_classification biology Temperature alpha-Glucosidases Maltose Hydrogen-Ion Concentration biology.organism_classification Molecular biology Culex quinquefasciatus Molecular Weight Culex Kinetics Enzyme chemistry Biochemistry Recombinant DNA biology.protein Insect Proteins Acarbose Oligopeptides Trisaccharides Biotechnology Plasmids |
| Zdroj: | Protein expression and purification. 110 |
| ISSN: | 1096-0279 |
| Popis: | 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 (kcat/Km) of the purified rCQMalIHis for maltotriose is higher than those for sucrose, maltotetraose, maltose and p-nitrophenyl-α-glucoside, indicating that the enzyme prefers maltotriose. Additionally, the rCQMalIHis is significantly inhibited by d-gluconic acid δ-lactone, but not by Mg(2+), Ca(2+) and EDTA. The rCQMalIHis is strongly inhibited by acarbose with IC50 67.8±5.6nM, but weakly inhibited by glucose with IC50 115.9±7.3mM. |
| Databáze: | OpenAIRE |
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