High yield overexpression and characterization of human recombinant proapolipoprotein A-I.
Human apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) where it defines the particle structure and stability and functions as the main activator of the enzyme lecithin:cholesterol acyltransferase (LCAT). ApoA-I is expressed in the liver as a preproprotein that is targeted to the endoplasmic reticulum for secretion; in plasma, an unknown protease removes the six amino acid long propeptide. In this study, the cDNA coding the human proapoA-I was cloned into an Escherichia coli vector; the overexpressed protein was purified to 99% homogeneity and was extensively characterized together with mature apoA-I purified from plasma. SDS-PAGE, mass spectrometry, and Edman sequence analysis showed that the initial Met residue needed for translation in E. coli is posttranslationally removed from the N-terminal sequence of the proapoA-I. The structural and functional analyses were carried out on the lipid-free and the lipid-bound proteins. ProapoA-I self associated, interacted with dimyristoyl phosphatidylcholine vesicles, and formed secondary structures very similar to the lipid-free apoA-I. Reconstituted HDL particles made with two initial molar ratios of palmitoyloleoyl phosphatidylcholine/cholesterol/apolipoprotein/Na-cholate had identical particle sizes and distributions when apoA-I or proapoA-I were used. Particles having diameters of 79 A and 98 A, containing two apoA-I or proapoA-I molecules per particle, were isolated and characterized. The particles contained the same amounts of alpha-helical structure, had very similar fluorescence properties, and activated LCAT equally well. We conclude that proapoA-I expressed and purified from E. coli is functionally and structurally indistinguishable from mature apoA-I purified from plasma when analyzed in vitro. Therefore, this recombinant proapoA-I and mutants derived from it will be important sources of protein for analyzing apoA-I structure and function, as well as for studies of proapoA-I processing.
McGuire
K A
KA
Department of Biochemistry, University of Illinois at Urbana-Champaign 61801, USA.
Davidson
W S
WS
Jonas
A
A
D016632:Apolipoprotein A-I / Q000737:chemistry*; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry; D013058:Mass Spectrometry; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D017421:Sequence Analysis; D013050:Spectrometry, Fluorescence
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D011498:Protein Precursors; D011994:Recombinant Proteins; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D004134:Dimyristoylphosphatidylcholine