胃幽門螺旋桿菌之麩胺基硫亞精胺合成酶(HP0233)的表現質體建構及功能分析

Abstract

The deduced amino acid sequence of the HP0233 gene shares more than 20% sequence identity with most glutathionylspermidine synthetase from several species, such as E. coli. The HP0233 open reading frame (1173 bp) was cloned into the pQE30 vector and over-expressed in Escherichia coli strain SG13009. The resulting N-terminally 6xHis-tagged HP0233 protein was purified by Ni–NTA affinity chromatography at a yield of 20 mg/L of bacteria culture. The recombinant HP0233 protein was used to produce polyclonal antibodies which could recognize single band of protein in H. pylori at different grown conditions by Western blotting analysis. Mass spectrometry and analytical ultracentrifugation have shown that the recombinant HP0233 protein exists as a monomer in solution. The major secondary structure was stable in the range of pH between 5.0 and 10.0. Thermal unfolding transition showed Tm valve was about 53 °C by circular dichroism spectroscopy. Results from functional assays showed that HP0233 possessed ATPase activity in reaction buffer containing 5 mM MgCl2 at pH 8.0 and 37 °C. The highest ATPase activity of HP0233 protein appeared in reaction buffer at pH 8.0. Reduced enzyme activity was observed in Hp0233 protein after stored at 45 °C for 20 min and assayed at 37 °C. The kinetic parameters of the recombinant HP0233 for the ATPase activity have been determined to have the apparent Km value of 0.48±0.16 mM for ATP substrate and a kcat value of 1.34±0.13 min-1. At 5 mM of Co2+, Mn2+, Fe2+, and Ca2+ can substitute for magnesium in Mg2+-dependent ATPase, but they were not able to significantly contribute to the enzymatic activity. ADP inhibition at 0.5-3.0 mM would suppress ATPase activity of HP0233 protein. The synthetase activity of HP0233 protein was not yet detected significantly. The endogenous HP0233 will be collected from H. pylori lysate with antibodies and the assay condition for synthetase activity needs to be further optimized. In vivo, HP0233 expression was reduced when H. pylori in acidic environment.