Expression, Purification, and Characterization of Recombinant Drosophila Choline Acetyltransferase

Donghai Wu; Norbert Schormann; Wei Lian; Johann Deisenhofer; Louis B. Hersh

doi:10.1111/j.1471-4159.1993.tb13635.x

Expression, Purification, and Characterization of Recombinant Drosophila Choline Acetyltransferase

Donghai Wu, Norbert Schormann, Wei Lian, Johann Deisenhofer, Louis B. Hersh

Research output: Contribution to journal › Article › peer-review

4 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	1416-1422
Number of pages	7
Journal	Journal of Neurochemistry
Volume	61
Issue number	4
DOIs	https://doi.org/10.1111/j.1471-4159.1993.tb13635.x
State	Published - Oct 1993

Keywords

Drosophila choline acetyltransferase
Enzyme crystallization
Enzyme physicochemical characterization
Recombinant enzyme

ASJC Scopus subject areas

Biochemistry
Cellular and Molecular Neuroscience

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10.1111/j.1471-4159.1993.tb13635.x

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title = "Expression, Purification, and Characterization of Recombinant Drosophila Choline Acetyltransferase",

abstract = "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.",

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author = "Donghai Wu and Norbert Schormann and Wei Lian and Johann Deisenhofer and Hersh, {Louis B.}",

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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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Expression, Purification, and Characterization of Recombinant Drosophila Choline Acetyltransferase

Abstract

Keywords

ASJC Scopus subject areas

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