TY - JOUR
T1 - Expression, Purification, and Characterization of Recombinant Drosophila Choline Acetyltransferase
AU - Wu, Donghai
AU - Schormann, Norbert
AU - Lian, Wei
AU - Deisenhofer, Johann
AU - Hersh, Louis B.
PY - 1993/10
Y1 - 1993/10
N2 - Abstract: 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 μmol/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 μ‐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.
AB - Abstract: 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 μmol/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 μ‐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.
KW - Drosophila choline acetyltransferase
KW - Enzyme crystallization
KW - Enzyme physicochemical characterization
KW - Recombinant enzyme
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U2 - 10.1111/j.1471-4159.1993.tb13635.x
DO - 10.1111/j.1471-4159.1993.tb13635.x
M3 - Article
C2 - 8376995
AN - SCOPUS:0027443452
SN - 0022-3042
VL - 61
SP - 1416
EP - 1422
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
IS - 4
ER -