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Temperature‐gradient gel electrophoresis for analysis and screening of thermostable proteases
Author(s) -
Sättler Andrea,
Riesner Detlev
Publication year - 1993
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501401122
Subject(s) - pmsf , subtilisin , phenylmethylsulfonyl fluoride , serine protease , chemistry , diisopropyl fluorophosphate , proteases , protease , temperature gradient gel electrophoresis , gel electrophoresis , polyacrylamide gel electrophoresis , chromatography , denaturation (fissile materials) , serine , active site , biochemistry , enzyme , nuclear chemistry , 16s ribosomal rna , gene
The thermal unfolding of microbial serine proteases was studied by temperature‐gradient gel electrophoresis (TGGE). Conditions for a native polyacrylamide gel electrophoresis were established, and the temperature gradient was applied perpendicularly to the direction of electrophoretic migration. Mobility changes of the protease molecules were indicative for thermally induced conformational changes. The transition temperature was determined with good accuracy. The native and active protease conformation was detected by an activity assay in the temperature‐gradient gel. As a consequence of the typical protease‐autoproteolytic reaction at elevated temperatures, the unfolded protease conformation could not be detected for non‐inhibited, active subtilisin. After inhibition by phenylmethylsulfonyl fluoride (PMSF) the complete structural transition could be followed by TGGE. This transition is “discontinuous”, 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 inhibitor diisopropyl fluorophosphate (DEP) led to two conformations at low temperature. One conformation is stabilized by 8°C, the other by at least 20°C as compared with PMSF inhibition. The influence of calcium ions on the subtilisin stability was investigated by a series of TGGE under different calcium concentrations. The strong calcium binding site is occupied even without added calcium, occupation of the weak binding site leads to a stabilization of 10°C with a binding constant around 10 6 M −1 . The subtilisin Carlsberg stability could also be investigated in unpurified bacterial culture supernatants. Thus, the method is suitable for screening of thermostable subtilisin mutants directly after expression in a bacterial host. For screening purposes TGGE was modified to a parallel form, which allows investigation of a series of samples in one and the same gel.