TEMPERATURE EFFECTS ON REFOLDING AND AGGREGATION OF A LARGE MULTIMERIC PROTEIN USING CAPILLARY-ZONE-ELECTROPHORESIS

Capillary zone electrophoresis (CZE) equipped with laser-induced fluor escence detection (LIFD) allows rapid and sensitive identification of folding and aggregation intermediates of P22 tailspike endorhamnosidas e using the intrinsic tryptophan fluorescence. Conversion of the protr imer into the native tailspike protein, which is the rate-limiting ste p, can be quenched at 0 degrees C and results in a loss of the protrim er in the refolding pathway. Results obtained from CZE-LIFD and polyac rylamide gel electrophoresis indicate the loss of folding intermediate s through the adsorption of polypeptide chains onto the wall of the re folding vial. Early aggregation intermediates are studied using a temp erature-sensitive folding tailspike mutant which shifts productive fol ding to the aggregation pathway at restrictive temperatures. By monito ring refolding intermediates during the temperature shift experiments, the early monomeric intermediate is identified as the thermolabile ju nctional intermediate between the productive and aggregation pathways. After passing the stage for accumulation of thermolabile intermediate , an increase in refolding temperature from 10 to 35 degrees C is carr ied out for achieving the optimal refolding kinetics and yields of tai lspike endorhamnosidase.