Expression of the C-terminal domain of human apolipoprotein A-I using a chimeric apolipoprotein

Protein Expression and Purification
Daniel E SalleePaul M Weers

Abstract

Human apolipoprotein A-I (apoA-I) is the most abundant protein in high-density lipoprotein, an anti-atherogenic lipid-protein complex responsible for reverse cholesterol transport. The protein is composed of an N-terminal helix bundle domain, and a small C-terminal (CT) domain. To facilitate study of CT-apoA-I, a novel strategy was employed to produce this small domain in a bacterial expression system. A protein construct was designed of insect apolipophorin III (apoLp-III) and residues 179-243 of apoA-I, with a unique methionine residue positioned between the two proteins and an N-terminal His-tag to facilitate purification. The chimera was expressed in E. coli, purified by Ni-affinity chromatography, and cleaved by cyanogen bromide. SDS-PAGE revealed the presence of three proteins with masses of 7 kDa (CT-apoA-I), 18 kDa (apoLp-III), and a minor 26 kDa band of uncleaved chimera. The digest was reloaded on the Ni-affinity column to bind apoLp-III and uncleaved chimera, while CT-apoA-I was washed from the column and collected. Alternatively, CT-apoA-I was isolated from the digest by reversed-phase HPLC. CT-apoA-I was α-helical, highly effective in solubilizing phospholipid vesicles and disaggregating LPS micelles. However, CT-a...Continue Reading

References

Jan 22, 1991·Biochemistry·D R BreiterH M Holden
Mar 10, 1995·The Journal of Biological Chemistry·H H SchmidtJ M Hoeg
Nov 26, 1996·Proceedings of the National Academy of Sciences of the United States of America·W Sean DavidsonA Jonas
Jan 21, 1997·Biochimica Et Biophysica Acta·J BergeronY L Marcel
Mar 3, 1998·Biochemical and Biophysical Research Communications·J L SoulagesM A Wells
Aug 26, 1998·Biochimica Et Biophysica Acta·Paul M WeersRobert O Ryan
Aug 14, 1999·Biochimica Et Biophysica Acta·Marc NiereAndreas Wiesner
Jan 31, 2002·Proceedings of the National Academy of Sciences of the United States of America·Jianjun WangRobert O Ryan
Jul 24, 2002·Protein Expression and Purification·Stacey E PanagotopulosW Sean Davidson
Dec 10, 2002·Biochemical and Biophysical Research Communications·Elda FavariLaura Calabresi
Jan 3, 2003·Protein Expression and Purification·Robert O RyanMichael N Oda
May 20, 2003·Nature Structural Biology·Michael N OdaJohn C Voss
Sep 23, 2009·Comparative Biochemistry and Physiology. Part A, Molecular & Integrative Physiology·V NarayanaswamiPaul M Weers
Oct 24, 2009·Proceedings of the National Academy of Sciences of the United States of America·Palaniappan Sevugan ChettyMichael C Phillips
Mar 15, 2011·Nature Structural & Molecular Biology·Rong HuangW Sean Davidson
Aug 16, 2011·Biochimica Et Biophysica Acta·Nicholas N LyssenkoMichael C Phillips
Aug 30, 2011·Protein Expression and Purification·David S Waugh
Oct 19, 2012·Arteriosclerosis, Thrombosis, and Vascular Biology·Mary G Sorci-Thomas, Michael J Thomas
Jul 31, 2014·The Journal of Biological Chemistry·Michael C Phillips
Jun 7, 2015·Archives of Medical Research·Xiaohu Mei, David Atkinson

Related Concepts

Protein Domain
Alkalescens-Dispar Group
Hybrid Proteins, Recombinant
Gene Expression
Pro-Apolipoprotein A-I
Chromatography, Affinity
High Pressure Liquid Chromatography Procedure
Cyanogen Bromide
Escherichia coli
Insecta

Related Feeds

ApoE, Lipids & Cholesterol

Serum cholesterol, triglycerides, apolipoprotein B (APOB)-containing lipoproteins (very low-density lipoprotein (VLDL), immediate-density lipoprotein (IDL), and low-density lipoprotein (LDL), lipoprotein A (LPA)) and the total cholesterol/high-density lipoprotein (HDL) cholesterol ratio are all connected in diseases. Here is the latest research.