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 sy stem. 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 bacte rial culture yielded 16-20 mg of purified recombinant ApoC-II. Sequenc ing and mass spectrometric analyses indicated that the isolated recomb inant ApoC-II contained predominantly (64%) the native form with threo nine as the N-terminus, but also contained a minor (36%) molecular for m of ApoC-II with an additional methionine at the N-terminus (Met-ApoC -II), Analysis of the recombinant ApoC-II by tryptic digestion and hig h performance liquid chromatography-electrospray mass spectrometry pro vides additional conclusive evidence that, with the exception of the N -terminus of Met-ApoC-II, the expressed ApoC-II has the expected pepti de 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 acti vation of bovine milk lipoprotein lipase. Thus, the recombinant ApoC-I I prepared from E. coli may have a pharmacological application for the treatment of patients with genetic hypertriglyceridemia caused by Apo C-II deficiency.