Expression, purification and characterization of the recombinant kringle 2 and kringle 3 domains of human plasminogen and analysis of their binding affinity for ω‐aminocarboxylic acids

Abstract

The kringle 2 (E161T/C162S/EEE[K2,,,/C169S]TT) and the kringle 3 (TYQ[K3,,]DS) domains of human plasminogen (HPg) were expressed in Eschericlzia coli in an expression vector with the phage T5 promotor/operator element N250PSN250P29 and the cDNA sequence for a hexahistidine tail to facilitate the isolation of the recombinant protein. A coagulation factor Xa (FXa)-sensitive cleavage site was introduced to remove the N-terminal histidine tag. In r-K2, mutations E161T and C162S were introduced to enhance the FXa cleavage yield and C169S to replace the cysteine residue, participating in the inter-kringle disulfide bridge between kringles 2 and 3. Recombinant proteins were isolated by affinity chromatography on Ni"-nitrilotriacetic acid/agarose and refolded under denaturing and reducing conditions followed by a non-denaturing and oxidising environment. The free thiol group in position 297 in r-K3 was selectively alkylated with iodoacetamide. The hexahistidine tail was successfully removed with FXa. The N-terminal sequence, the amino acid composition and the molecular mass analyses are in agreement with the expected data. The correct arrangement of the disulfide bonds was verified by sequence analysis of the corresponding thermolytic and subtilisin fragments. r-K2 exhibits weak binding to lysine-Bio-Gel. The weak binding affinity of r-K2 for o-aminocarboxylic acids is confirmed by intrinsic fluorescence titration with 6-aminohexanoic acid (NH,C,COOH) indicating a Kd of approximately 401 pM. In contrast, r-K3 seems to be devoid of a binding affinity for w-aminocarboxylic acids. Considering earlier determined Kd values of kringle 1, kringle 4 and kringle 5, the binding affinity of HPg kringle domains for NH,C,COOH is proposed to decrease in the following order, kringle 1 > kringle 4 > kringle 5 > kringle 2 > knngle 3.Plasminogen is the main component of the fibrinolytic system and its activated form, plasmin, is responsible for the proteolytic degradation of fibrin in blood clots. Human plasminogen (HPg) is a single-chain proenzyme of 791 amino acids. The sequence has been established by amino acid sequence analysis [l-31 and

Kringle 2 Mediates High Affinity Binding of Plasminogen to an Internal Sequence in Streptococcal Surface Protein PAM

“…Two lysine-containing 13-amino acidlong repeated sequences (a1 and a2) located in the surfaceexposed NH 2 terminus of PAM mediate the interaction with Pg (22,23). In this paper we show that K2, although binding very weakly to free lysine or lysine analogues (13,14), mediates high affinity binding of Pg to PAM and its internal a repeats. …”

“…The proteolytic fragments K1-K3, K4, and mini-plasminogen (m-Pg), which encompasses K5 and the serine proteinase domain, were obtained by partial cleavage of human Pg by elastase and purified as described (27). Construction, expression, and purification of rK1(SE[K1]D) has been previously described (12) (13). rK2 and rK3 were expressed with a NH 2 -terminal hexahistidine (His 6 ) tag to facilitate purification.…”

“…K2 binds weakly to lysine or 6-AHA (13,14). Nevertheless, involvement of the lysine-binding site of K2 in the interaction with the a1a2 repeat region was suggested by the demonstration that 6-AHA completely inhibited the binding of 125 I-labeled VEK30 to rK2 (Fig.…”

“…Lysine analogues, such as 6-aminohexanoic acid (6-AHA), mimic COOH-terminal lysines in the interaction with kringles and the structural basis of the interactions between some kringles, particularly K1 and K4, and 6-AHA has been disclosed (9 -12). The affinity of the different kringles for lysine or 6-AHA is variable, such that K1 displays the highest affinity, whereas K3 shows no measurable interaction with 6-AHA (13,14). Some kringles, notably K1 and K5, also bind lysine analogues and proteins lacking a carboxylgroup.…”

Mutational Analysis of Affinity and Selectivity of Kringle-Tetranectin Interaction

“…Cys-169 and Cys-299, which in Plg forms an inter-domain disulfide bridge between kringles 2 and 3, were substituted by Ser in the rPlg K2 and rPlg K3 constructs, respectively, to prevent refolding problems and aggregation, an approach that has also been applied earlier (37). The four kringle derivatives were then expressed, refolded, and immobilized on sensorchips, and binding analysis of rTN and rTN T149Y was performed.…”