Title
Mycobacterium tuberculosis H37Ra: a surrogate for the expression of conserved, multimeric proteins of M.tb H37Rv
Author
Vishant Mahendra Boradia, Pravinkumar Patil, Anushri Agnihotri, Ajay Kumar, Kalpesh Kumar Rajwadi, Ankit Sahu, Naveen Bhagath, Navdeep Sheokand, Manoj Kumar, Himanshu Malhotra, Rachita Patkar, Navi Hasan, Manoj Raje, Chaaya Iyengar Raje
Year
2016
Journal
Microbial Cell Factories
Abstract
Obtaining sufficient quantities of recombinant M.tb proteins using traditional approaches is often unsuccessful. Several enzymes of the glycolytic cycle are known to be multifunctional, however relatively few enzymes from M.tb H37Rv have been characterized in the context of their enzymatic and pleiotropic roles. One of the primary reasons is the difficulty in obtaining sufficient amounts of functionally active protein. In the current study, using M.tb glyceraldehyde-3-phosphate dehydrogenase (GAPDH) we demonstrate that expression in E. coli or M. smegmatis results in insolubility and improper subcellular localization. In addition, expression of such conserved multisubunit proteins poses the problem of heteromerization with host homologues. Importantly the expression host dramatically affected the yield and functionality of GAPDH in terms of both enzymatic activity and moonlighting function (transferrin binding). The applicability of this system was further confirmed using two additional enzymes i.e. M.tb Pyruvate kinase and Enolase. Our studies establish that the attenuated strain M.tb H37Ra is a suitable host for the expression of highly hydrophobic, conserved, multimeric proteins of M.tb H37Rv. Significantly, this expression host overcomes the limitations of E. coli and M. smegmatis expression and yields recombinant protein that is qualitatively superior to that obtained by traditional methods. The current study highlights the fact that protein functionality (which is an an essential requirement for all in vitro assays and drug development) may be altered by the choice of expression host.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Biochemistry