Lee C.,Daegu University |
Kim J.,Daegu University |
Hong S.,Daegu University |
Goo B.,Daegu University |
And 2 more authors.
Applied Biochemistry and Biotechnology | Year: 2013
A gene coding for the extracellular esterase (EstK) was cloned from the psychrotrophic bacterium Pseudomonas mandelii based on its partial amino acid sequence as determined by mass spectrometry. The entire open reading frame consisting of 1,011 bp was expressed in Escherichia coli as a soluble protein and purified by nickel-chelated affinity chromatography and Capto Q column chromatography. Here, we show that the 33-kDa recombinant EstK protein (rEstKsp) had a substrate preference for esters of short-chain fatty acids, especially, p-nitrophenyl acetate. Optimum activity of rEstKsp was at pH 8.5 and 40 C. The esterase activity remained similar from a range of 4∼20 C, but the maximum activity varied depending upon pH. With p-nitrophenyl acetate as the substrate, K M was 210 μM and k cat was 3.4 s-1. Circular dichroism and fluorescence spectroscopy results revealed that rEstKsp had a predominantly α-helical structure and maintained its folded state at 4∼40 C. Interestingly, the tertiary structure of rEstKsp was predicted based on the structures of other hyperthermophilic esterases. Our results demonstrated that both native and rEstKsp are active at low temperatures and have a unique substrate preference for p-nitrophenyl acetate. © 2012 Springer Science+Business Media New York. Source
Kim J.-W.,Daegu University |
Kim K.,Daegu University |
Ahn Y.,Gyeongsan Science High School |
Jeong H.,Gyeongsan Science High School |
And 5 more authors.
BMB Reports | Year: 2010
Exendin-4 (Ex-4), a peptide secreted from the salivary glands of the Gila monster lizard, can increase pancreatic β-cell growth and insulin secretion by activating glucagon-like peptide-1 receptor. In this study, we expressed a fusion protein consisting of exendin-4 and the human immunoglobulin heavy chain (Ex-4/IgG-Fc) in E. coli and explored its potential therapeutic use for the treatment of insulin-resistant type 2 diabetes. Here, we show that the Ex-4/IgG-Fc fusion protein induces expression of insulin receptor substrate-2 in rat insulinoma INS-1 cells. Our findings therefore suggest that Ex-4/IgG-Fc overexpressed in E. coli could be used as a potential, long-acting glucagon-like peptide-1 mimetic. Source