Bacterial Expression of Biologically Active High Molecular Weight Kininogen Light Chain

Abstract

SummaryHuman high molecular weight kininogen (HK), a single chain plasma glycoprotein, serves as a cofactor in the contact system of blood coagulation. After cleavage by human plasma kallikrein, the nonapeptide bradykinin is released. The HK light chain (LC) contains coagulant activity, which requires both the ability to bind the contact system zymogens, prekallikrein and factor XI, and the ability to interact with negatively charged surfaces. Since bacterial expression might not be successful if carbohydrate was required for activity, we evaluated that possibility by incubating plasma HK with endoglycosydase F. Although the procedure removed detectable N-linked carbohydrate, no change in specific activity occurred. We then developed a bacterial expression system to produce recombinant HK LC. The cDNA coding for the HK LC was prepared by polymerase chain reaction (PCR), digested with restriction enzymes EcoRI and PstI, and introduced into the bacterial expression vector pKK223-3. E. coli harboring this recombinant plasmid (pSKl) expressed HK LC upon induction with isopropylthio-galactoside (IPTG). The recombinant protein (27 kDa), when transferred onto a PVDF membrane, was recognized by monospecific polyclonal anti-HK LC-antibodies. The recombinant HK LC was purified by heparin agarose affinity chromatography to homogeneity and found to have a specific activity of 28 coagulant units per mg protein, similar to the specific activity of the LC derived by proteolytic digestion of human plasma HK. We conclude: 1) The HK LC synthesized in bacteria is biologically active, and 2) the 40% carbohydrate content of the HK LC is not required for its cofactor activity.

Inhibition of Platelet Adhesion and Aggregation by a Defined Region (Gly-486–Lys-502) of High Molecular Weight Kininogen

“…The resulting HKa was composed of two bands of 62 and 46 kDa when analyzed by reduced SDS-gel electrophoresis. Glutathione S-transferase (GST) fused to domains 3, 5, and 6 of HK or to sequences derived from domain 5 were produced as previously described (37). GST was amino-terminally attached to the following sequences of HK: Gly-235-Met-357 (domain 3), Lys-420 -Ser-513 (domain 5), and Thr-503-Ser-626 (domain 6) as well as Lys-420 -Asp-474, and His-475-Lys-502 (amino-terminal and carboxyl-terminal domain 5 sequences).…”

A Novel Antithrombotic Role for High Molecular Weight Kininogen as Inhibitor of Plasminogen Activator Inhibitor-1 Function

“…HK was digested with plasma kallikrein (HK:kallikrein, 100:1, mol/mol) for 20 min at 37°C to obtain HKa, which appears as two bands of 62 and 46 kDa when analyzed by reduced SDS-gel electrophoresis. Glutathione S-transferase (GST) fused to domain 3, 5, or 6 of HK or to sequences derived from domain 5 were produced as described previously (37 …”

Structural requirements for cathepsin B and cathepsin H inhibition by kininogens

“…The recombinant proteins were purified on a glutathione affinity column as described earlier (Kunapuli et al, 1993). The fractions containing the protein were identified by the absorption at 280/xm.…”