Abstract
BackgroundObtaining 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.ResultsIn 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0537-0) contains supplementary material, which is available to authorized users.
Highlights
Obtaining sufficient quantities of recombinant M.tb proteins using traditional approaches is often unsuccessful
Subcellular localization of rGAPDH Recombinant M.tb glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was expressed in three different strains viz. E. coli, M. smegmatis and M. tuberculosis H37Ra, protein purified from these host strains are referred to as EC-H-GAPDH, MS-H-GAPDH, MT-H-GAPDH, MS-GAPDH-H, MT-GAPDH-H to reflect the expression host, position of Histidine tag and protein
The presence of GAPDH was confirmed with α-GAPDH [9] and α-His antibodies to confirm the presence of rGAPDH-His (Fig. 1a; Additional file 3: Table 1)
Summary
Obtaining sufficient quantities of recombinant M.tb proteins using traditional approaches is often unsuccessful. Several laboratories are involved in identifying Mycobacterium tuberculosis (M.tb) proteins that play a crucial role in immunomuodulation, gene regulation, pathogenesis and virulence with the aim of developing novel anti-mycobacterials. Functional characterization of these activities requires large quantities of purified protein and commonly, recombinant M.tb proteins are expressed in available E. coli strains. Due to their high GC content and intrinsic hydrophobicity the products are often functionally inactive. We demonstrated that in pathogenic M.tb H37Rv, GAPDH and other conserved proteins function
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