Expression, Purification, Crystallization And Its Physical And Chemical Characteristics Of Esterase Gene From Mycobacterium Tuberculosis

Mycobacterium tuberculosis, the pathogenic microorganism of the Infectious tuberculosis is harmful and a serious threat of the humanbeing. It has attracted scientist’s attention and concern for a long time. And a lot of researches have done on it. With the emergence of Drug-resistant strains of Mycobacterium tuberculosis mutations, the vast majority of conventional TB drugs have emerged in varying degrees of resistance, which result the tuberculosis has a rebound in the global trend. The number of tuberculosis patients in China increases 1.45 million every year, which makes the TB becomes the first contagious disease killer in China. Studies have shown that its pathogenicity and tolerance are closely related of the highly esters ratio of its composition. And esters metabolism are mainly controled by esterases. To study the structure and function of esterase contribute to a better understanding of Mycobacterium tuberculosis. Thus, we can develop a new generation of tuberculosis targeted drugs.In this paper, the enzyme rv3036c gene which we studyed is from Mycobacterium tuberculosis H37Rv. However, due to the lack of relevant knowledge such as structure and signal pathways of Rv3036c, the functions and role of Rv3036c in Mycobacterium tuberculosis ester metabolism is still unclear.This work mainly includes the construction of the rv3036c gene recombinant vector, the recombinant protein’s expression, purification, crystallization and to study the physical and chemical properties.Recombinant rv3036c gene in Escherichia coli are include the expression of recombinant Rv3036c protein. Followed by the using the Ni-NTA affinity chromatography, DEAE ion exchange chromatography and Superdex 75 gel chromatography methods for the protein’s separation and purification,we have got the more than 99% protein of Rv3036c. SDS-PAGE electrophoresis showed that its molecular weight is 25.5KD. By spectrophotometric verify its esterase activity.Using the hanging drop methord of vapor diffusion we screen and optimize the crystallization conditions of Rv3036c. Ultimately we obtained a single crystal under a crystallization condition for suitable X-ray diffraction.These studies established the effective way of expression purification, protein crystallization,determined esterase of Rv3036c, and have done some useful attempts for the further study on the structure based drag resistance and pathogenicity mechanism of Mycobacterium tuberculosisin. The results of our study provided important information for the anti-tuberculosis drug design and development, have important theoretical significance and practical application value.