Expression, purification, and characterization of human and rat acetyl coenzyme A carboxylase (ACC) isozymes.
Acetyl coenzyme A (acetyl-CoA) carboxylase isozyme 1 (ACC1) and acetyl-CoA carboxylase isozyme 2 (ACC2) are critical for de novo fatty acid synthesis and for the regulation of beta-oxidation. Emerging evidence indicates that one or both isozymes might be therapeutic targets for the treatment of obesity, type 2 diabetes, and dyslipidemia. One of the major obstacles in the field is the lack of readily-available source of recombinant human ACC enzymes to support systematic drug discovery efforts. Here, we describe an efficient and optimal protocol for expressing and isolating recombinant mammalian ACCs with high yield and purity. The resultant human ACC2, human ACC1, and rat ACC2 possess high specific activities, are properly biotinylated, and exhibit kinetic parameters very similar to the native ACC enzymes. We believe that the current study paves a road to a systematic approach for drug design revolving around the ACC inhibition mechanism.
Cheng
Dong
D
Department of Obesity and Metabolic Research, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, PO Box 5400, Princeton, NJ 08543-5400, USA. dong.cheng@bms.com
Chu
Ching-Hsuen
CH
Chen
Luping
L
Feder
John N
JN
Mintier
Gabe A
GA
Wu
Yuli
Y
Cook
Joseph W
JW
Harpel
Mark R
MR
Locke
Gregory A
GA
An
Yongmi
Y
Tamura
James K
JK
D000103:Acetyl-CoA Carboxylase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae; D002846:Chromatography, Affinity; D019343:Citric Acid / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D019809:Streptavidin / Q000737:chemistry
D007527:Isoenzymes; D011994:Recombinant Proteins; D019343:Citric Acid; D008316:Malonyl Coenzyme A; D019809:Streptavidin; D000103:Acetyl-CoA Carboxylase