A Novel Method for Expression and Purification of the C‐Terminal Domain of Apolipoprotein A‐I

Abstract

Apolipoprotein A‐I (apoA‐I) is the most abundant protein in high‐density lipoprotein, an anti‐antherogenic complex responsible for reverse cholesterol transport. Human apoA‐I is a 243 amino acid protein comprised of two domains, a helix bundle N‐terminal domain, and an unstructured C‐terminal (CT) domain comprising residues 179‐243 which adopts a helical conformation when lipid bound. To better understand the function of the CT apoA‐I, a novel strategy was employed to produce large quantities of this small fragment using a recombinant expression system. The strategy required the introduction of a methionine residue in front of the CT domain to be able to cleave with cyanogen bromide and isolate the desired fragment. However, apoA‐I contains three methionine residues making it difficult to cleave and isolate the correct fragment. Therefore, a new construct was engineered by combining CT apoA‐I with apolipophorin III (apoLp‐III), an insect apolipoprotein that lacks methionine. A unique methionine was then introduced by site‐directed mutagenesis between apoLp‐III and CT apoA‐I. The chimeric protein was expressed in E. coli BL21 cells, purified by nickel affinity chromatography, and cleaved by cyanogen bromide. SDS‐PAGE revealed the presence of three distinct bands at 7 kDa (CT apoA‐I), 18 kDa (apoLp‐III), and a minor band at 26 kDa of uncleaved protein. Then, reversed‐phase HPLC was employed to isolate the 7 kDa fragment. SDS‐PAGE of the first peak eluted from the C8 column confirmed isolation of CT apoA‐I. This novel expression system allows for production of significant quantities of CT apoA‐I needed for the analysis to understand how this important part of apoA‐I functions.This project was supported by NIH/NIGMS GM089564 and GM008074.