Jensen, P.K., King, J. and Lee, C.S. (1998) Analytical Chemistry, 70, 730-736.
Investigating temperature effects on refolding and aggregation of a large multimeric protein using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) equipped with laser-induced fluorescence detection (LIFD) allows rapid and sensitive identification of folding and aggregation intermediates of P22 tailspike endorhamnosidase using the intrinsic tryptophan fluorescence. Conversion of the protrimer into the native tailspike protein, which is the rate-limiting step, can be quenched at 0oC and results in a loss of the protrimer in the reolfing pathway. Results obtained from CZE-LIFD and polyacrylamide gel electrophoresis indicate the loss of folding intermediates through the adsorption of polypeptide chains onto the wall of the refolding vial. Early aggregation intermediates are studied using a temperature-sensitive folding tailspike mutant which shifs productive folding to the aggregation pathway at restrictive temperatures. By monitoring refolding intermediates during the temperature shift experiments, the early monomeric intermediate is identified as the thermolabile junctional intermediate between the productive and aggregation pathways. After passing the stage for accumulation of thermolabile intermediate, an increase in refolding temperature from 10 to 35oC is carried out for achieving for optimal refolding kinetics and yields of tailspike endorhamnosidase.


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