(43) Assay Card

General Information
Summary Cyclisation of beta lactamase enhanced protein stability.
Condition The stability against heat precipitation was quantitatively tested as follows. Two-hundred ul of a 40 uM protein solution, directly taken from the ligation reaction in the reaction buffer (200 mM NaCl, 200 mM NaPi, 50 mM MENSA, pH 5.0) was incubated at various temperatures (from 40 to 70¬C). The insoluble fraction was removed by centrifugation at 20800óor 30 min. The protein pellets and the supernatant, precipitated with 5% TCA, were dissolved with sodium dodecyl sulfate (SDS) loading buffer. Each soluble and insoluble fraction was analyzed with SDS-polyacrylamide gel electrophoresis (PAGE). After Coomassie brilliant blue staining, the individual bands were quantified with a densitometer (Model 300A, Molecular Dynamics).
Result As the midpoint of the transition (Tm), we obtained 45¬C for the linear form of BLA-S and 50¬C for the circular form of BLA-S under these conditions, indicating a stability enhancement of 5¬C by cyclization.
AssayType Temperature

Iwai H, Pluckthun A (1999) Circular beta-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett 459:166-72

Proteins Assayed beta lactamase - BLA