title: Expression, purification, and characterization of human acetyl-CoA carboxylase 2.
abstract: The full-length human acetyl-CoA carboxylase 1 (ACC1) was expressed and purified to homogeneity by two separate groups (Y.G. Gu, M. Weitzberg, R.F. Clark, X. Xu, Q. Li, T. Zhang, T.M. Hansen, G. Liu, Z. Xin, X. Wang, T. McNally, H. Camp, B.A. Beutel, H.I. Sham, Synthesis and structure-activity relationships of N-{3-[2-(4-alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy derivatives as selective acetyl-CoA carboxylase 2 inhibitors, J. Med. Chem. 49 (2006) 3770-3773; D. Cheng, C.H. Chu, L. Chen, J.N. Feder, G.A. Mintier, Y. Wu, J.W. Cook, M.R. Harpel, G.A. Locke, Y. An, J.K. Tamura, Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes, Protein Expr. Purif., in press). However, neither group was successful in expressing the full-length ACC2 due to issues of solubility and expression levels. The two versions of recombinant human ACC2 in these reports are either truncated (lacking 1-148 aa) or have the N-terminal 275 aa replaced with the corresponding ACC1 region (1-133 aa). Despite the fact that ACC activity was observed in both cases, these constructs are not ideal because the N-terminal region of ACC2 could be important for the correct folding of the catalytic domains. Here, we report the high level expression and purification of full-length human ACC2 that lacks only the N-terminal membrane attachment sequence (1-20 and 1-26 aa, respectively) in Trichoplusia ni cells. In addition, we developed a sensitive HPLC assay to analyze the kinetic parameters of the recombinant enzyme. The recombinant enzyme is a soluble protein and has a K(m) value of 2 microM for acetyl-CoA, almost 30-fold lower than that reported for the truncated human ACC2. Our recombinant enzyme also has a lower K(m) value for ATP (K(m)=52 microM). Although this difference could be ascribed to different assay conditions, our data suggest that the longer human ACC2 produced in our system may have higher affinities for the substrates and could be more similar to the native enzyme.
authors.affiliation: Department of Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
authors.forename: Ki Won
authors.initials: KW
authors.lastname: Kim
authors.forename: Harvey
authors.initials: H
authors.lastname: Yamane
authors.forename: James
authors.initials: J
authors.lastname: Zondlo
authors.forename: James
authors.initials: J
authors.lastname: Busby
authors.forename: Minghan
authors.initials: M
authors.lastname: Wang
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005663:Furans / Q000494:pharmacology; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010169:Palmitic Acids / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D012995:Solubility; D018411:Spodoptera / Q000166:cytology / Q000235:genetics
chemical_list: D004791:Enzyme Inhibitors; D005663:Furans; D010169:Palmitic Acids; D011994:Recombinant Proteins; D006639:Histidine; C014289:5-(tetradecyloxy)-2-furancarboxylic acid; C056166:MEDICA 16; C520105:ACACB protein, human; D000103:Acetyl-CoA Carboxylase