EXPRESSION OF A RECOMBINANT KRINGLE-V OF HUMAN APOLIPOPROTEIN(A) - ANTIBODY CHARACTERIZATION AND SPECIES-SPECIFICITY

Lipoprotein(a) is a macromolecular complex consisting of a low-density lipoprotein-like particle with an additional glycoprotein, apolipopro tein(a) [apo(a)], linked to apolipoprotein B-100 via a disulfide bond. Apo(a) is highly homologous to plasminogen. We have cloned the sequen ce corresponding to the kringle V domain of apo(a) from human liver cD NA using an experimental approach involving use of the polymerase chai n reaction. The protein product of this clone was expressed in the cyt oplasmic compartment of Escherichia coli as a MalE fusion protein. Fus ion apo(a) Kr V was isolated from cytoplasmic extracts and purified by amylose-agarose affinity chromatography by eluting with 10 mM maltose . The fusion protein was injected into sheep in order to generate a po lyclonal anti-apo(a) Kr V antibody. The antibody raised reacted agains t both reduced Lp(a) and the C-terminal domain of apo(a), correspondin g to a sequence extending from Iir 33 to the C-terminal residue, but d id not react with the N-terminal domain containing the repeated Kr IV sequences. The presence of the Kr V sequence was detected in every hum an apo(a) size isoform tested but only in apo(a) from human and chimpa nzee among a panel of apo(a) proteins derived from different animal sp ecies. (C) 1996 Academic Press, Inc.