Lactate dehydrogenase from

1.25 µg of Lactate dehydrogenase from rabbit muscle (Sigma-Aldrich®, Sigma, USA) was denatured with 1 molar guanidine hydrochloride (GdnHCl) during 10 min at 25 °C. After that, 100 mM of NaH₂PO₄ pH 7 buffer was added, the final volume mix was 500 µl. The mix was incubated at 25 °C for 90 min with 287 nM chaperones: BSA (negative control), OppAWT, OppA mutants and HtrA (as positive control). Again, The SIGMAFAST™ (Sigma, USA) Protease Inhibitor was added according to the protocol of the commercial kit. Lactate dehydrogenase (71.43 nM) activity assays were performed at 25 °C according to Sigma-Aldrich® (Sigma, USA) protocol. β-NADH was measured by spectrophotometry at 340 nm every 10 s for 5 min. LDH units in each assay were calculated according to LDH activity assay protocol from Sigma-Aldrich® (Sigma, USA).
The molar ratio beween LDH and chaperone-like proteins (OppA, BSA and HtrA) was set to the same proportion as before, 1 to 4, while GdnHCl was applied at a 20 mM concentration because at this concentration level the LDH activity remains unaltered, all of which had been empirically determined prior to our bioassays.

Test proteins–Carbonic Anhydrase from Bovine erythrocytes (BCA), Chicken egg white lysozyme, Ovalbumin from egg white, Malic Dehydrogenase (Mitochondrial) from Porcine Heart (MDH) and Lactate dehydrogenase from Pig muscle (LDH) were purchased from Sigma; guanidine hydrochloride was purchased from Sigma. 70S ribosome from E.coli MRE600 was prepared as described earlier [15 (link)–17 (link)]. Plasmids containing RRF, EFG and IF3 genes under T7 promoters [18 (link)] were kindly provided by Prof. Umesh Varshney, IISc, India. E.coli (pKR15) cells used for isolation and purification of tRNA^Glu were a kind gift from Dr. Jack Lapointe, University of Laval, Quebec, Canada. GTP, its non-hydrolyzable analogue GMPPNP, FITC (Fluorescein-5-isothiocyanate), Isopropyl-β-D-thiogalactopyranoside (IPTG), DEAE cellulose and the antibiotic fusidic acid were purchased from Sigma. BIOGEL P-60 and P-100 gel medium were purchased from BIORAD. The synthetic deca-peptide VGDANPALQK was a kind gift from Prof. D.K.Chattoraj, NIH, USA. E.coliLJ14 (temperature sensitive strain for RRF) was a kind gift from Dr. Santanu Dasgupta, Uppsala University, Sweden.