EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT DROSOPHILA CHOLINE-ACETYLTRANSFERASE

A cDNA for Drosophila choline acetyltransferase (EC 2.3.1.6; ChAT) was fused with a polyhistidine sequence and expressed in Escherichia coli . The recombinant enzyme was purified to a specific activity of 500 mu mol/min/mg of protein using metal affinity chromatography and ion exch ange chromatography. Kinetic properties of the recombinant enzyme did not differ significantly from those previously determined. Circular di chroism (CD) spectra revealed that the secondary structure of the enzy me is largely alpha-helical. Intrinsic fluorescence spectra of the enz yme indicate that its tryptophan residues are buried. Neither CD nor f luorescence spectra changed significantly in the presence of substrate s. The cysteine content of the recombinant Drosophila ChAT was determi ned to be 16 in the absence and 22 in the presence of 6 M guanidine hy drochloride. Finally, crystallization of recombinant Drosophila ChAT w as achieved.