BACTERIAL EXPRESSION AND CHARACTERIZATION OF HUMAN RECOMBINANT APOLIPOPROTEIN(A) KRINGLE-IV TYPE-9

Elevated plasma lipoprotein(a) [Lp(a)] is an independent risk factor f or several vascular diseases. Lp(a) particles are generated through th e formation of a disulfide bond between Cys(4057) of kringle TV type 9 , (KIVt9), of the multikringle apolipoprotein(a) [apo(a)] and a cystei ne in apoB-100 low-density Lipoprotein (LDL). To better understand thi s interaction, we have expressed and purified KIVt9 from Escherichia c oli as a His-Tag fusion protein. Dithiothreitol (DTT)-treated purified KIVt9 migrated as a single similar to 17.3-kDa band on SDS-PAGE gels. Without DTT, an additional band twice the molecular weight of KIVt9 w as observed. The double-size band presumably resulted hom dimerization of individual kringles, through their unpaired cysteine residues, sin ce a mutation Cys(4057) -->Ser ([Ser(4057)]KIVt9) abolished dimer form ation. Using a gel-shift assay, we showed that KIVt9 could couple to 1 4-amino-acid apoB-100 synthetic peptides (apoB(3732-3745) and apoB(431 9-4332)) containing Cys(3734) or cys(4326). Both Of these apoB-100 cys teines have been reported to associate with apo(a) to generate Lp(a). In the presence of either apoB-100 peptide, KIVt9 was shifted to a hig her molecular weight that was consistent with the covalent addition of a 1.2-kDa apoE-100 peptide. Identical apoB-100 peptides in which the cysteine residues were replaced by alanine ([Ala(3734)]apoB(3732)-(374 5) and [Ala(4326)]apo(B4319-4332)) had no effect in the gel-shift assa y. Furthermore, [Ser(4057)]KIVt9 did not covalently interact with apoB (3732-3745) or apoB(4319-4332) These results indicated that KIVt9 coup les to the Cys-apoB-100 peptides through a disulfide linkage. This sys tem may be suitable for further investigating the apo(a)/apoB-100 coup ling reaction and the structure of KIVt9 through X-ray crystallographi c studies. (C) 1998 Academic Press.