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R. Gupta, O.P. Srivastava; Effect of Site-Directed Mutagenesis (Deamidation of N78 and N146 and N78/N146) on Chaperone Activity of B-Crystallin . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2362.
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Purpose: The objective of the study was to determine the effect of deamidation in αB-crystallin on chaperone activity by using site-specific mutagenesis (N78D, N146D and N78D/N146D) . Methods: Three αB-crystallin mutants (i.e., N78D, N146D and N78D/N146D) were generated. The mutations at the desired sites were confirmed by DNA sequencing. Recombinant native αB-crystallin and the three mutated αB species were expressed using IPTG, and each species was purified to homogeneity using ion-exchange chromatography followed by size-exclusion HPLC. The chaperone activities of the purified recombinant native and mutated αB-crystallin species were determined by the heat-induced aggregation of citrate synthase (CS). Results: DNA sequencing data confirmed the site-specific mutations at desired sites (i.e., at N78D, N146D and N78D/N146D). Following purification, the recombinant native and mutated proteins showed a single protein band on SDS-PAGE. The mutated crystallins (N78D and N146D) at concentration of 0.1 mg showed 15-20% suppression of CS aggregation compared to the native recombinant αB crystallin. At an identical concentration, the crystallin with double mutation (N78D/N146D) showed up to 40% suppression of CS aggregation suggesting that deamidation resulted in a partial loss of chaperone activity. Conclusions: The results suggested that deamidation in mutated crystallins (i.e., N78D, N146D and N78D/N146D) caused a partial loss of chaperone activity compared to native αB-crystallin.
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