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 mumol/min/mg of protein using metal affinity chromatography and ion exchange chromatography. Kinetic properties of the recombinant enzyme did not differ significantly from those previously determined. Circular dichroism (CD) spectra revealed that the secondary structure of the enzyme is largely alpha-helical. Intrinsic fluorescence spectra of the enzyme indicate that its tryptophan residues are buried. Neither CD nor fluorescence spectra changed significantly in the presence of substrates. The cysteine content of the recombinant Drosophila ChAT was determined to be 16 in the absence and 22 in the presence of 6 M guanidine hydrochloride. Finally, crystallization of recombinant Drosophila ChAT was achieved.
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas.
D
D
Wu
N
N
Schormann
W
W
Lian
J
J
Deisenhofer
L B
LB
Hersh
D000818:Animals; D001483:Base Sequence; D002795:Choline O-Acetyltransferase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D003460:Crystallization; D004247:DNA / Q000235:genetics; D004330:Drosophila / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D004247:DNA; D002795:Choline O-Acetyltransferase