Chimeric apolipoproteins as novel plasma cholesterol lowering agents (1002.2)

Abstract

Apolipoprotein E3 (apoE3, 299 residues, 34 kDa) and apolipoprotein A‐I (apoA‐I, 243 residues, 28 kDa) play significant roles in regulating plasma cholesterol levels, by virtue of their abilities to facilitate cholesterol uptake by binding the low density lipoprotein receptor (LDLr) and promoting cholesterol efflux from macrophages, respectively. We aim to generate a chimeric apolipoprotein by combining the LDLr binding region in the N‐terminal domain (NT) of apoE3 (residues 1‐191) with the cholesterol efflux‐promoting segment in the C‐terminal domain (CT) of apoA‐I (residues 181‐243). Overlap extension PCR was employed to fuse the nucleotide sequence encoding apoE3 NT with apoA‐I CT, followed by ligation into a pET20b(+) vector. The DNA sequence of the apoE3‐NT/apoA‐I‐CT chimera was verified. Overexpression of the chimera in E. coli yielded ~40 mg/L of purified protein. SDS‐PAGE and Western blot analyses confirmed the presence of a protein band of ~31 kDa, and MALDI‐TOF mass spectrometry revealed a mass of 30,977 Da (expected mass: 30,975 Da). Preliminary circular dichroism data of the chimera revealed α‐helical character, similar to apoE3 and apoA‐I. Further biophysical, spectroscopic and, lipid‐, LDLr‐binding and cholesterol efflux functional assays will be performed. The chimera has the potential to be used as a powerful cholesterol‐lowering agent to protect against cardiovascular disease.Grant Funding Source: Supported by the NIH #GM105561, #GM089564, and MARC grant T34GM008074‐22