Journal article
High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli
Journal of lipid research, Vol.53(8), pp.1708-1715
08/2012
DOI: 10.1194/jlr.D028043
PMCID: PMC3540857
PMID: 22636422
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.
Details
- Title: Subtitle
- High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli
- Creators
- Loren E. Smith - University of CincinnatiJun Yang - University of CincinnatiLeah Goodman - the Children's Hospital Oakland Research Institute, Oakland, CA 94609Xinqi Huang - the Children's Hospital Oakland Research Institute, Oakland, CA 94609Rong Huang - University of CincinnatiJames Dressman - University of CincinnatiJamie Morris - University of CincinnatiR. A. Gangani D. Silva - University of CincinnatiW. Sean Davidson - University of CincinnatiGiorgio Cavigiolio - the Children's Hospital Oakland Research Institute, Oakland, CA 94609
- Resource Type
- Journal article
- Publication Details
- Journal of lipid research, Vol.53(8), pp.1708-1715
- DOI
- 10.1194/jlr.D028043
- PMID
- 22636422
- PMCID
- PMC3540857
- NLM abbreviation
- J Lipid Res
- ISSN
- 0022-2275
- eISSN
- 1539-7262
- Publisher
- Elsevier Inc
- Number of pages
- 8
- Language
- English
- Date published
- 08/2012
- Academic Unit
- Anesthesia
- Record Identifier
- 9984949238402771
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