Purification of ACAT Protein

Abstract

Acyl‐coenyzme A:cholesterol acyltransferase (ACAT‐1) is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholestryl esters from cholesterol and long‐chain fatty acyl coenzyme A. Recently, we employed human embryonic kidney 293 (HEK293) cells to stably express the recombinant human ACAT‐1 protein bearing a six‐hisitidine tag at the N‐terminus and a Flag‐tag at the C‐terminus. The incorporation of the six–histidine and flag‐tag into the ACAT‐1 sequence allows the ACAT‐1 fusion protein to be purified using two affinity columns. This enzyme was purified by solubilizing the cell membrane with 1M of potassium chloride and 3‐[(3‐cholidopropyl)dimethylammonio]‐1‐propansulfanate (CHAPS). The lysate was then loaded onto an immobilize nickel column followed by a monoclonal Flag antibody column. The flag column allowed us to elute the fusion protein by the flag peptide. More importantly, the elution of the fusion protein using the Flag peptide is one of the most critical steps in the purification because it eliminates the use of low pH (pH 3 to pH4) elution conditions that were used in earlier purifications using ACAT monoclonal antibodies. This new purification scheme allows us to recover a larger quantity of active ACAT‐1 protein for further study of the structure and function of the human ACAT‐1 protein. Supported by NIH T34GM008411