Mycobacterium tuberculosis H37Ra: a surrogate for the expression of conserved, multimeric proteins of M.tb H37Rv

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.

Mycobacterium tuberculosis Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Functions as a Receptor for Human Lactoferrin

“…The full length M.tb GAPDH gene was cloned and expressed in M.tb H37Ra to obtain recombinant wild type GAPDH (wt rGAPDH) as described previously (Boradia et al, 2016 ). During plasmid screening two point mutations were detected (i) an Arginine to Serine mutation at position 142 (N142S) and (ii) a Proline to Leucine mutation at position 295 (P295L).…”

“…During plasmid screening two point mutations were detected (i) an Arginine to Serine mutation at position 142 (N142S) and (ii) a Proline to Leucine mutation at position 295 (P295L). Each plasmid was individually transformed into M.tb H37Ra using standard methods (Wards and Collins, 1996 ), recombinant proteins were purified as described previously (Boradia et al, 2016 ), the mutant proteins are referred to as rGAPDH(N142S) and rGAPDH(P295L) respectively.…”

“…Both mutant forms of the protein were expressed and purified as described previously for wt rGAPDH (Boradia et al, 2016 ). Purification was confirmed by western blotting, detection was done after incubation with Mouse anti-His (Sigma) (1:3,000) for 1 h followed by incubation with anti-Mouse IgG HRP (Sigma) (1:8,000) for 1 h. The enzyme activity of wt rGAPDH, rGAPDH(N142S), and rGAPDH(P295L) purified from cytosol fraction was studied by measuring the increase in the absorbance at 340 nm due to oxidative reduction of NAD + to NADH.…”

Repurposing ethyl bromopyruvate as a broad-spectrum antibacterial

“…Recombinant GAPDH (rGAPDH) was purified from Mtb H37Ra as described previously. 9 The purified enzyme was dialysed against 50 mM Tris (pH 8.0) and 150 mM NaCl and enzyme activity of rGAPDH was assayed. Briefly, 500 ng of purified enzyme was added to 100 lL of assay buffer (50 mM HEPES, 10 mM sodium arsenate and 5 mM EDTA, pH 8.5), 1 mM NAD !…”

“…Recombinant pyruvate kinase (PykA) was purified from strain Mtb H37Ra, which over-expresses this protein as described. 9 Briefly, PykA activity was assayed using a lactate dehydrogenase-coupled assay that measures the decrease in absorbance at 340 nm resulting from the oxidation of NADH. To determine activity of Mtb PykA, a reaction mixture containing 50 mM HEPES, pH 7.5, 10 mM MgCl 2 , 50 mM KCl, 2 mM ADP, 10 mM sodium phosphoenol pyruvate, 0.3 mM NADH and 1.5 U of lactate dehydrogenase was pre-incubated at 25 C for 5 min.…”