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Expression, Purification, and Characterization of Recombinant Drosophila Choline Acetyltransferase
Author(s) -
Wu Donghai,
Schormann Norbert,
Lian Wei,
Deisenhofer Johann,
Hersh Louis B.
Publication year - 1993
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1993.tb13635.x
Subject(s) - recombinant dna , choline acetyltransferase , affinity chromatography , circular dichroism , microbiology and biotechnology , enzyme , biochemistry , complementary dna , escherichia coli , chemistry , enzyme assay , biology , cholinergic , gene , neuroscience
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.