کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020352 1542331 2015 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Functional expression and molecular characterization of Culex quinquefasciatus salivary α-glucosidase (MalI)
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Functional expression and molecular characterization of Culex quinquefasciatus salivary α-glucosidase (MalI)
چکیده انگلیسی
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 3.75 U/ml, under optimal culture conditions. The purified rCQMalIHis showed a single band of molecular weight of approximately 92 kDa on SDS-PAGE. After Endoglycosidase H digestion, a single band at 69 kDa was found on SDS-PAGE analysis, suggesting that rCQMalIHis is a glycoprotein. Additionally, tryptic digestion and LC-MALDI MS/MS analysis suggested that the 69 kDa 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 Mg2+, Ca2+ and EDTA. The rCQMalIHis is strongly inhibited by acarbose with IC50 67.8 ± 5.6 nM, but weakly inhibited by glucose with IC50 115.9 ± 7.3 mM.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 110, June 2015, Pages 145-150
نویسندگان
, , , , , ,