Identification of Critical Residues in c-di-GMP Receptor Clpxoo from Xanthomonas oryzae pv. oryzae

Abstract

Abstract: The purpose of this study is to identify the critical residues in Clpxoo of a c-di-GMP signal receptor in Xanthomonas oryzae pv. oryzae（Xoo）. By the gene mutation of amino acid site of Clpxoo protein，the construction of expressing vector，induced protein expressed，and analysis of Ni-NTA Resin affinity chromatography，the prokaryotic expression of Clpxoo and point mutants was conducted and the protein was purified.. The binding affinities of c-di-GMP with native and variant Clpxoo were measured by isothermal titration calorimetry（ITC）experiments. Under optimized conditions for protein expression and purification，the proteins of Clpxoo point mutants and point mutants of Clpxoo that does not bind with c-di-GMP were acquired successfully while using gene site-directed mutagenesis and bridging PCR. Results showed that site 70 of aspartic acids and site 99 of glutamic acid were found to be the key residues for their binding to c-di-GMP.

Key words: Xanthomonas oryzae pv. pryzae, c-di-GMP, signal receptor protein, point mutant, binding affinity

CLC Number:

10.13560/j.cnki.biotech.bull.1985.2016.12.020

LI Jian-yu,LI Bo,CHEN Hua-min,YANG Feng-huan,HE Chen-yang,TIAN Fang. Identification of Critical Residues in c-di-GMP Receptor Clpxoo from Xanthomonas oryzae pv. oryzae[J]. Biotechnology Bulletin, 2016, 32(12): 124-129.

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