Expression, purification, and characterization of human acetyl-CoA carboxylase 2
| Autor: | Minghan Wang, Harvey Yamane, James Busby, Ki Won Kim, James Zondlo |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Stereochemistry
Molecular Sequence Data Gene Expression Palmitic Acids Spodoptera Biology Sensitivity and Specificity Isozyme law.invention Inhibitory Concentration 50 Hplc assay law Acetyl-coenzyme A carboxylase Enzyme Stability Trichoplusia Animals Humans Histidine Amino Acid Sequence Enzyme Inhibitors Furans Cells Cultured Chromatography High Pressure Liquid chemistry.chemical_classification Dose-Response Relationship Drug Acetyl-CoA carboxylase biology.organism_classification Recombinant Proteins Pyruvate carboxylase Molecular Weight Kinetics Enzyme Solubility Biochemistry chemistry Recombinant DNA Baculoviridae Acetyl-CoA Carboxylase Biotechnology |
| Zdroj: | Protein Expression and Purification. 53:16-23 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2006.11.021 |
| Popis: | 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 μM 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 μM). 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. |
| Databáze: | OpenAIRE |
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