Two methods for large-scale purification of recombinant human choline acetyltransferase.
Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 micromol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.
Kim
Ae-Ri
AR
Department of Biochemistry, University of Western Ontario, London, Ont., Canada N6A 5C1.
Doherty-Kirby
Amanda
A
Lajoie
Gilles
G
Rylett
R Jane
RJ
Shilton
Brian H
BH
D001483:Base Sequence; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D002795:Choline O-Acetyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D002686:Chitin; D006639:Histidine; D002795:Choline O-Acetyltransferase