ANNOUNCEMENTS
The aim of the major project was the overexpression and purification of the recombinant protein GmhC from the gram-negative bacteria Helicobacter pylori (H. pylori). H. pylori is a causative agent of peptic ulcer and sores in the stomach and small intestine of humans and other primates. The heptose sugar is crucial for maintaining the outer membrane integrity of lipopolysaccharide (LPS), and this in turn protects the gram-negative bacteria from outer environment modifications and host’s immune response. The protein GmhC is a bifunctional enzyme which acts at first and third step of the heptose biosynthesis pathway, which is in turn a part of LPS biosynthesis pathway, predominantly present in the gram-negative bacteria. The recombinant plasmid containing the synthesized gene of interest, gmhC, incorporated in the lac operon of pET 28a (+) vector cassette was procured. Competent cells for creating transformed colonies of bacteria possessing the plasmid pET 28a (+) were prepared. Confirmation of successful transformation was achieved by plasmid isolation, followed by gel electrophoresis to visualize the plasmid DNA. Induction process was set up to over express the protein at various concentrations of an inducer, which is a molecular mimic of allolactose, called the isopropyl thio-beta galactoside (IPTG), with varying temperature conditions. It was observed that protein got over expressed most efficiently at 0.01mM concentration of IPTG upon overnight incubation at 18o temperature. The purification of protein was done through Ni-NTA chromatography, since the gene of interest contains a hexa-his-tag at the C-terminal.. However, in order to achieve purification with homogeneity, anion exchange chromatography was performed. Further, Gel Exclusion chromatography may be performed so as to set up crystallization trials by hanging drop and sitting drop method.
