High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli.
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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ABSTRACT: ApoA-II is the second most abundant protein on HDL making up ∼20% of the total protein but its functions have still only been partially characterized. Recent methodological improvements have allowed for the recombinant expression and characterization of human apoA-II which shares only 55% sequence homology with murine apoA-II. Here we describe the purification of the two most common polymorphic variants of apoA-II found in inbred mouse strains, differing at 3 amino acid sites. C57BL/6 mice having variant apoA-II(a) have lower plasma HDL levels than FVB/N mice that have variant apoA-II(b). Characterization of the helical structure of these two variants reveals a more alpha-helical structure for the FVB/N apoA-II. These changes do not alter the lipid or HDL binding of the two apoA-II variants, but significantly increase the ability of the FVB/N variant to promote both ABCA1 and ABCG1 mediated cellular cholesterol efflux. These differences may be differentially altering plasma HDL apoA-II levels. In vivo, neither C57 nor FVB apoA-II protein levels are affected by the absence of apoE, while an apoE/apoA-I double deficiency results in a 50% decrease of plasma FVB apoA-II but results in undetectable levels of C57 apoA-II in the plasma. FVB apoA-II is able to form an HDL particle in the absence of apoE or apoA-I.PLoS ONE 01/2014; 9(2):e88705. · 3.73 Impact Factor
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ABSTRACT: Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy "footprinting" assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops.Protein Expression and Purification 07/2013; · 1.43 Impact Factor