Figure 2.

Kinetic analysis of the reactions of HaloTag-Cutinase (HC) fusions and linker molecules. A) The HC fusion protein was treated with a ligand to block either the cutinase (left) or HaloTag (right) domain and then treated with the linker for specified times before treating with an excess of an inhibitor-fluorophore conjugate to label the remaining unreacted protein. The yields for each reaction were determined by monitoring the loss of the starting protein by gel electrophoresis and the data were fit to determine the first order rate constant for the reaction. B) This experiment was repeated to measure the rate constant for the coupling of two fusion proteins, as described in the text.