Cloning, Expression, Purification and Biological Activity Analysis of Recombinant Helicobacter pylori FabI as a Drug Target

Abstract

Helicobacter pylori (H. pylori) is an infectious agent colonized in gastric epithelium and leads to serious diseases such as ulcers and gastric carcinoma. H. pylori infection requires rapid and effective treatment options however existing therapies gradually diminish in efficacy due to the development of resistance. Type II fatty acid synthesis (FAS-II) pathway is a potent target for drug discovery studies because of its absence in humans and vital necessity for bacteria. In the last step of the synthesis, trans-2-enoyl-ACP is reduced to acyl-ACP with cofactor of NADH by enoyl-ACP reductase, FabI. In this study, recombinant HpFabI was successfully produced using an aLICator ligation-independent cloning and expression vector system for the first time. HpFabI gene was cloned, and then expressed, and the protein was purified in high yield. Recombinant HpFabI with a molecular mass of similar to 30 kDa was confirmed with Western Blot analysis and its concentration was determined in the range of 1.406-3.9495 mg/ml by Bradford Assay. The enzyme-specific activity of HpFabI was determined as 1.5871 nmol min(-1) mu g(-1) by using NADH and crotonoyl-CoA as cofactor and substrate, respectively. HpFabI was produced in high yield to facilitate future inhibition studies including high throughput screening studies for FabI inhibition to contribute novel drug development studies fighting against H. pylori infection.