Cloning and in-silico characterization of MmaA1 protein of mycobacterium tuberculosis
Student name: Ms Vasundhara Sharma Guide: Dr Chaithanya Madhurantakam Year of completion: 2018 Host Organisation: TERI School of Advanced Studies Abstract: Mycobacterium tuberculosis (Mtb) H37Rv is a pathogenic bacteria which causes tuberculosis. The pathogenecity of this bacteria is related to its cell wall. Mtb infection remains the major health problem which has led to high demands of antitubercular drugs. The comimg up of MDR (Multidrug resistance) and XDR (Extensively drug resistant) has made the situation more severe. The cell wall of Mtb consists majorly of the mycolic acids. Varieties of genetic determinants have been identified which play essential role in the synthesis pathway of the mycolic acid. Among these, MmaA1 plays an essential role of conversion of cis olefin to trans olefin at the proximal end. In the present study, we have used pUC57 as cloning vector with our gene of interest, mmaa1. The gene fragment was transferred to an expression vector pET28a+. Ligation protocol was performed between the backbone of pET28a+ and the mmaA1 gene fragment from pUC57 cloning vector. Further, in-silico characterization of MmaA1 was done and a refined MmaA1 model was developed through homology modelling. The putative 3D structure was validated usingPSVS. Multiple sequence alignment and evolutionary relationship analysis were derived between MmaA1 and its similar family proteins like MmaA2, MmaA3 and MmaA4. Secondary structure prediction was done using PHYRE2 server and a topology diagram was created for MmaA1 protein. In addition, putative catalytic site for the MmaA1 protein were identified and docking studies were done with the common drug molecules used as first line of antibiotics for tuberculosis treatment like isoniazid and pyrazineamide.