Construction and Characterization of Helicobacter pylori tRNA-guanine transglycosylase (TGT) in pET28a His-tagged vector
The tgt gene encodes for tRNA-guanine transglycosylase (TGT), the key enzyme to the incorporation of the modified nucleoside queuine (7-[[4, 5-cis-dihydroxy-l-cyclopenten- 3-yl] amino] methyl)-7 -deazaguanosine) and archaeine (7 -amidino-7 deazaguanine) into tRNA. Queuine, for example, is exchanged for guanine in the wobble position (number 34) of tRNAs with the anticodon sequence GUN. These tRNAs code for tyrosine, histidine, asparagine, and aspartate. This incorporation, which appears to be unique in its mechanism, has several postulated but few proven physiological roles. Helicobacter pylori, in addition to having the tgt gene and the modified nucleoside incorporation, is an extremely prevalent bacterial infection in the world today. Unfortunately, very few studies have accurately characterized the infection. Many of the studies of the Helicobacter pylori TGT have been hampered by the difficulty of isolating and purifying active enzytne. By sub-cloning the H. pylori tgt gene into pET28a, a commercially available vector that appends an amino- and/or carboxyl- terminus His-Tag to sub cloned proteins, we hope to generate a more efficient system for the preparation of H. pylori TGT. The H. pylori tgt gene (strain G27) was obtained by polymerase chain reaction with specific primers. This was sub cloned into pET28a and confirmed by DNA sequencing (University of Michigan, Medical School DNA Sequencing Core Facility).