Cloning and Expression of a Haloacid Dehalogenase Enzyme

Abstract

The purpose of this research project is to clone the gene JHP1130 from Helicobacter pylori (H. pylori). JHP1130 is a member of the Haloacid Dehalogenase (HAD) superfamily, which is characterized by a varied group including but not limited to phosphatases, epimerases, and dehalogenases (Allen). JHP1130 has been identified as a phosphatase, but not much is known about its physiological role. In order to learn more about this role, the gene was cloned into a pet21b vector for subsequent expression. Once a clone was created, the plasmid was transformed into BL21 E. coli cells for protein expression. After induction, confirmation of protein expression was verified by checking the protein against a molecular weight ladder. The expected molecular weight of the desired protein was roughly 25 kDa and a band at this molecular weight confirmed the presence of the correct protein. The next phase of testing will involve purifying the protein as well as testing various substrates capable of binding to the enzyme. The importance of this research project lies with the fact that JHP1130 has little homology to the phosphatases of other types of bacteria. If the reason behind this difference is that H. pylori possesses a different metabolic pathway from most bacteria, then this research project could reveal a treatment for H. pylori infection that won't have adverse effects on the normal bacterial flora existing within the human body.