Abstract
Background: Mycobacterial α-crystallin (Acr) is a chaperone that prevents misfolding of proteins when Mycobacterium tuberculosis is found in a latent form in the host tissue. Methods: Using insulin as a model substrate and utilizing polynomial graphs, we attempted to predict molecular-level interactions that are a function of the oligomeric state of the recombinant protein. The chaperone activity of the recombinant oligomeric Acr was measured at 60°C with Acr samples obtained before gel filtration chromatography and compared with a gel-filtered sample. Results: The polynomial graphs constructed showed improved molecular coverage of the insulin B chain by the oligomer. The 2 nd order coefficient is the one that changes with the oligomeric ratio of Acr and improves chaperone activity. Polynomial analysis suggested that it could be a useful parameter to predict chaperone activity for potential in vitro batches of M. tuberculosis Acr based on the dynamic nature of the association and disassociation of oligomers. Conclusions: The results showed that coverage of insulin B chain improved with higher ratio of 9-mer as compared to lower ratios. This was shown by both simulation plots and actual assay data. The polynomial graphs showed increase in the 2 nd order coefficient, thus suggesting the important role of oligomerisation in improved molecular coverage of insulin B chain.
Highlights
Mycobacterial α-crystallin (Acr) is an important protein in latent tuberculosis (TB)
Expression of the recombinant acr was initially done at 50 ml scale, using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) induction (37°C for 3h)
Expression and activity studies The His-tag purification and gel filtration showed greater than 95% purity as seen in the gel analysis along with upper molecular weight bands (Figure 1a)
Summary
Mycobacterial α-crystallin (Acr) is an important protein in latent tuberculosis (TB). Acr is active inoligomeric state, and though it is known to act as a trimer of trimers (Chang et al, 1996) or a dodecamer (Kennaway et al, 2005; Panda et al, 2017), yet it is difficult to study the mechanism by which it inhibits the thermal aggregation or dithiothreitol (DTT)-induced aggregation of insulin. Author’s response: Suggested change Acr is active in oligomeric state, and though it is known to act as a trimer of trimers (Chang et al, 1996) or a dodecamer (Kennaway et al, 2005; Panda et al, 2017), yet it is difficult to study the mechanism by which it inhibits the thermal aggregation or dithiothreitol (DTT)-induced aggregation of insulin. The polynomial graphs showed increase in the 2nd order coefficient, suggesting the important role of oligomerisation in improved molecular coverage of insulin B chain
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