TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS FOR ANALYSIS AND SCREENING OF THERMOSTABLE PROTEASES

The thermal unfolding of microbial serine proteases was studied by tem perature-gradient gel electrophoresis (TGGE). Conditions for a native polyacrylamide gel electrophoresis were established, and the temperatu re gradient was applied perpendicularly to the direction of electropho retic migration. Mobility changes of the protease molecules were indic ative for thermally induced conformational changes. The transition tem perature was determined with good accuracy. The native and active prot ease conformation was detected by an activity assay in the temperature -gradient gel. As a consequence of the typical protease-autoproteolyti c reaction at elevated temperatures, the unfolded protease conformatio n could not be detected for non-inhibited, active subtilisin. After in hibition by phenylmethylsulfonyl fluoride (PMSF) the complete structur al transition could be followed by TGGE. This transition is ''disconti nuous'', i.e. the thermal transition is either very slow, compared to the time of electrophoresis, or irreversible, as known for subtilisins from calorimetric data. Inhibition by the strong serine specific inhi bitor diisopropyl fluorophosphate (DFP) led to two conformations at lo w temperature. One conformation is stabilized by 8-degrees-C, the othe r by at least 20-degrees-C as compared with PMSF inhibition. The influ ence of calcium ions on the subtilisin stability was investigated by a series of TGGE under different calcium concentrations. The strong cal cium binding site is occupied even without added calcium, occupation o f the weak binding site leads to a stabilization of 10-degrees-C with a binding constant around 10(6) m-1. The subtilisin Carlsberg stabilit y could also be investigated in unpurified bacterial culture supernata nts. Thus, the method is suitable for screening of thermostable subtil isin mutants directly after expression in a bacterial host. For screen ing purposes TGGE was modified to a parallel form, which allows invest igation of a series of samples in one and the same gel.