CLONING, EXPRESSION, AND CHARACTERIZATION OF HUMAN APOLIPOPROTEIN(A) KRINGLE IV37

A portion of kringle IV37 (KIV37) of apolipoprotein (a), (apo(a)), was polymerase chain reaction-cloned from human liver cDNA The protein pr oduct of this clone was expressed in Escherichia coli as a poly histid ine fusion protein. Based on recovery of purified fusion apo(a) KIV37 protein expression levels were estimated to be 10 mg/g of E. coli cell paste. Mass spectral analysis showed the molecular mass of fusion apo (a) KIV37 to be 12,260 +/- 1 daltons. Almost all fusion apo(a) KIV37 w as expressed as inclusion bodies and had to be refolded. Fusion apo(a) KIV37 was isolated from the inclusion bodies and purified by lysine-S epharose affinity chromatography by eluting with 0.2 M epsilon-aminoca proic acid. The fusion protein was treated with thrombin to yield a ho mogeneous, functional apo(a) KIV37 domain composed of 92 amino acids h aving a molecular mass of 10,510 +/- 1 daltons. N-terminal protein seq uencing and amino acid analysis have confirmed the sequence and compos ition of apo(a) KIV37. The molar extinction coefficient, epsilon, apo( a) KIV37 was determined to be 3.1. x 10(4) M(-1) cm(-1), and the pI wa s measured to be 6.7 +/- 0.1. In addition, the dissociation constants, K-d, for a series of 11 lysine analogs have been determined by measur ing the change in intrinsic fluorescence of apo(a) KIV37 upon saturabl e binding with these compounds. K-d values ranged from 4.2 +/- 0.9 mu M for trans-4-(aminomethyl)cyclohexanecarboxylic acid to 4.6 +/- 0.4 m M for L-arginine. Apo(a) KIV37 binds to plasmin-treated fibrinogen wit h an EC(50) value of 14 +/- 1.2 mu M and prevents the binding of Lp(a) to plasmin-treated fibrinogen with an IC50 value of 16 +/- 6 mu M. Lp (a) binds to the plasmin-treated fibrinogen surface-with an EC(50) val ue of approximately 1.0 +/- 0.3 nM. These studies demonstrate that apo (a) KIV37 Can be expressed at high levels, refolded properly, and used as a fully functional lysine-binding domain. In addition, these resul ts also demonstrate that apo(a) KIV37 provides the major interaction o f Lp(a) with fibrinogen. One additional weak binding site in Lp(a) is adequate to describe overall Lp(a) binding to fibrinogen.