Figure 4

Expression and purification of CatIBs. (a) The utilized protocol for IB isolation. (b) 12% SDS-polyacrylamide gels showing the different steps of IB isolation. After cell disruption using sonification TkAMPpase was expressed mainly in the insoluble fraction (IS) (2 µL of 1:10 dilution was used on the gel). Washing steps with Tris–HCl buffer were applied to decrease protein background (10 µL from each (W1, W2 and W3) was loaded on the SDS PAGE). The final IB preparation showed only minor impurities (IB) (2 µL of IB in a 1:16 dilution was loaded on the SDS-PAGE). (c) Specific activity of the CatIBs compared to the HIAs obtained from 60 °C heat treatment after 60 min was determined. The used reaction conditions were 2 mM substrate (NMP) and 50 mM potassium phosphate in MOPS buffer (pH 7) at 80 °C. Regular samples were taken at different time points over a period of 15 min. Standard deviation is derived from three experiments. M protein ladder, IS insoluble protein fraction, S soluble protein fraction. Cell disruption method: sonification.