Bacterial expression and characterization of mature apolipoprotein A-I

Stacey E. Panagotopulos, Scott R. Witting, Erica M. Horace, J. Nicholas Maiorano, W. Sean Davidson

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Plasma levels of apolipoprotein A-I (apoA-I) are correlated with reduced incidence of heart disease due to the critical role of this protein in reverse cholesterol transport. Because of its diversity of function and poorly understood structure, much research has sought to understand how the structure of apoA-I facilitates its function. A popular approach has been the use of site-directed mutagenesis followed by structural and functional studies. There are a wide variety of expression systems available to produce these mutant proteins including eukaryotic cell lines and prokaryotic cells such as Escherichia coli. Expression in a bacterial system is generally favorable because it can produce large amounts of pure protein quickly and economically through the use of affinity tags on the expressed protein. Unfortunately, many of these systems are not ideal for the production of apolipoproteins because, in many cases, the proteolytic digestion required to remove the affinity tag also cleaves the target protein. Here we describe a method that produces large amounts of recombinant protein that is easily purified using a histidine (His) affinity tag that is cleaved with IgA protease from Neisseria gonorrhoeae. This enzyme does not cleave the wild type apoA-I sequence, leaving intact, mature apoA-I (containing a Thr-Pro- on the N-terminus). We show that this recombinant protein is similar to wild type protein in structure and function using circular dichroism analysis, lipid clearance assays, recombinant particle formation and cholesterol efflux assays. This system is particularly useful for the bacterial production of apolipoproteins because of the extreme specificity of IgA protease for its target cleavage site.

Original languageEnglish (US)
Pages (from-to)353-361
Number of pages9
JournalProtein Expression and Purification
Volume25
Issue number2
DOIs
StatePublished - Nov 23 2002

ASJC Scopus subject areas

  • Biotechnology

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    Panagotopulos, S. E., Witting, S. R., Horace, E. M., Nicholas Maiorano, J., & Sean Davidson, W. (2002). Bacterial expression and characterization of mature apolipoprotein A-I. Protein Expression and Purification, 25(2), 353-361. https://doi.org/10.1016/S1046-5928(02)00020-7