Isolation and characterization of recombinant human apolipoprotein C-II expressed in Escherichia coli.
A full-length recombinant human apolipoprotein C-II (ApoC-II) has been successfully expressed in Escherichia coli using the T7 expression system. The recombinant ApoC-II. which was expressed intracellularly in the inclusion bodies, was solubilized with 8 M urea and purified using Sephadex G-75 gel permeation chromatography. Four liters of the bacterial culture yielded 16-20 mg of purified recombinant ApoC-II. Sequencing and mass spectrometric analyses indicated that the isolated recombinant ApoC-II contained predominantly (64%) the native form with threonine as the N-terminus, but also contained a minor (36%) molecular form of ApoC-II with an additional methionine at the N-terminus (Met-ApoC-II). Analysis of the recombinant ApoC-II by tryptic digestion and high performance liquid chromatography-electrospray mass spectrometry provides additional conclusive evidence that, with the exception of the N-terminus of Met-ApoC-II, the expressed ApoC-II has the expected peptide sequence. However, this extra N-terminal methionine residue can be excised by further in vitro treatment with methionine aminopeptidase. The purified recombinant ApoC-II was found to be competent in the activation of bovine milk lipoprotein lipase. Thus, the recombinant ApoC-II prepared from E. coli may have a pharmacological application for the treatment of patients with genetic hypertriglyceridemia caused by ApoC-II deficiency.
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Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA. chi-sun-wang@omrf.uokhsc.edu
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D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008071:Lipoprotein Lipase / Q000378:metabolism; D013058:Mass Spectrometry; D063208:Methionyl Aminopeptidases; D008892:Milk / Q000201:enzymology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D011994:Recombinant Proteins; D008071:Lipoprotein Lipase; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases