After purification on nickel-nitrilotriacetate acid (Ni-NTA) resin, the yield of cIFN-alpha was 400 mu g/ml cell-free reaction solution. The resultant cIFN-alpha was fully biologically active as demonstrated by its anti-cancer effect and immunoassay signals. (C) 2011 Elsevier Inc. All rights reserved.”
“Secreted

phospholipase A(2)s form a large family of proteins involved in diverse biological and pathophysiological processes. Group IIE secreted phospholipase A(2) (sPLA(2)-IIE) is one of the latest discovered members of this family. Previous studies revealed that the expression profile HKI-272 clinical trial of sPLA(2)-IIE was restricted to a few tissue types including brain, heart, lung and placenta compared to the broad expression profile of other isoforms. Accumulating evidence suggests that sPLA(2)-IIE might play a pivotal role in the progression of inflammatory processes. However, functional

study of sPLA(2)-IIE was hindered by the low yield of soluble expressed protein. In this study, we have expressed human and mouse sPLA(2)-IIE in Escherichia coli in the form of inclusion bodies. The inclusion bodies were dissolved, purified and refolded in a stepwise dialysis approach and further purified. We obtained soluble and active proteins for human and mouse sPLA(2)-IIE with a final yield of 1.1 and 1.2 mg/500 mL bacterial culture, respectively. The refolded sPLA(2)-IIEs find more exhibited similar calcium and pH dependence of their enzymatic activity with those expressed in COS cells. This protein expression and purification protocol will facilitate the further structural and functional studies of human and mouse sPLA(2)-IIEs. (C) 2011 Elsevier Inc. All rights reserved.”
“The gene encoding a carboxylesterase

from Anoxybacillus sp., PDF1, was cloned and sequenced. The recombinant protein was expressed in Escherichia coil BL21, under the control of isopropyl-beta-D-thiogalactopyranoside-inducible T7 promoter. The enzyme, designated as PDF1Est, was purified by heat shock and ion-exchange column chromatography. The molecular mass of the native protein, as determined by SDS-PAGE, was about 26 kDa. PDF1Est was active under a broad pH range (pH 5.0-10.0) and a broad temperature selleck compound range (25-90 degrees C), and it had an optimum pH of 8.0 and an optimum temperature of 60 degrees C. The enzyme was thermostable carboxylesterase, and did not lose any activity after 30 min of incubation at 60 degrees C. The enzyme exhibited a high level of activity with p-nitrophenyl butyrate with apparent K(m), V(max), and K(cat) values of 0.348 +/- 0.030 mM, 3725.8 U/mg, and 1500 +/- 54.50/s, respectively. The effect of some chemicals on the esterase activity indicated that Anoxybacillus sp. PDF1 produce an carboxylesterase having serine residue in active site and -SH groups in specific sites, which are required for its activity. (C) 2011 Elsevier Inc. All rights reserved.