Expression, isolation and characterization of a mutated human plasminogen kringle 3 with a functional lysine binding site

Each kringle of human plasminogen (HPg) except kringle 3 (K3) exhibits affi nity for omega-aminocarboxylic acids. Assuming that the K3 domain contains a preformed but nonfunctional lysine binding site (LBS), Lys(311) was alter ed by site-directed mutagenesis into Asp(311) in accordance with the consen sus sequence of the LBS. Cys(297) involved in the interkringle disulfide br idge was mutated into Ser(297) to minimize dimerization and aggregation. Th e mutated K3 TYQ[K3(HPg)/C297S/K311D]DS (r-K3(mut)) was expressed in Escher ichia coli, isolated on an Ni2+-nitrilotriacetic acid-agarose column, refol ded and purified on a lysine Bio-Gel column. Fluorescence titration indicat es affinity of r-K3(mut) for omega-aminocarboxylic acids with the following association constants (K-ass, mM(-1)): 5-aminopentanoic acid: 1.3; 6-amino hexanoic acid: 4.2; 7-aminoheptanoic acid: 0.5; trans-(aminomethyl)cyclohex anecarboxylic acid: 12.7;p-benzylaminesulfonic acid: 11.8. r-K3(mut) exhibi ts an affinity similar to native and mutated (R220G, E221D) K2. The results indicate the presence of a preformed but nonfunctional LBS in native K3 of HPg. We were able to demonstrate for the first time that an appropriate mu tation in the LBS of a kringle produced a weak but distinct affinity for om ega-aminocarboxylic acids.