Temperature-gradient gel electrophoresis for analysis and screening of thermostable proteases.
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 (DFP) led to two conformations at low temperature. One conformation is stabilized by 8 degrees C, the other by at least 20 degrees 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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Sättler
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Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
Riesner
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D002021:Buffers; D002118:Calcium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D010450:Endopeptidases / Q000032:analysis*; D004795:Enzyme Stability; D006358:Hot Temperature; D007531:Isoflurophate / Q000494:pharmacology; D009154:Mutation; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000032:analysis; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013381:Subtilisins / Q000032:analysis / Q000037:antagonists & inhibitors; D013696:Temperature
D002021:Buffers; D015842:Serine Proteinase Inhibitors; D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; D010450:Endopeptidases; D012697:Serine Endopeptidases; D013381:Subtilisins; D002118:Calcium