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  2. High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli

High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli

  • J Lipid Res. 2012 Aug;53(8):1708-15. doi: 10.1194/jlr.D028043.
Loren E Smith 1 Jun Yang Leah Goodman Xinqi Huang Rong Huang James Dressman Jamie Morris R A Gangani D Silva W Sean Davidson Giorgio Cavigiolio
Affiliations

Affiliation

  • 1 Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45273, USA.
Abstract

Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lack of knowledge about its structure, specific biological function, and role in Cardiovascular Disease. Here we present a novel Escherichia coli-based recombinant expression system that produces highly pure mature human apoA-II at substantial yields. A Mxe GyrA intein containing a chitin binding domain was fused at the C terminus of apoA-II. A 6× histidine-tag was also added at the fusion protein's C terminus. After rapid purification on a chitin column, intein auto-cleavage was induced under reducing conditions, releasing a peptide with only one extra N-terminal Met compared with the sequence of human mature apoA-II. A pass through a nickel chelating column removed any histidine-tagged residual fusion protein, leaving highly pure apoA-II. A variety of electrophoretic, mass spectrometric, and spectrophotometric analyses demonstrated that the recombinant form is comparable in structure to human plasma apoA-II. Similarly, recombinant apoA-II is comparable to the plasma form in its ability to bind and reorganize lipid and promote Cholesterol efflux from macrophages via the ATP binding cassette transporter A1. This system is ideal for producing large quantities of recombinant wild-type or mutant apoA-II for structural or functional studies.

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