Production and characterization of recombinant human proteinase inhibitor 6 expressed in Pichia pastoris

Abstract

The human intracellular serine proteinase inhibitor, proteinase inhibitor 6 (PI-6), was expressed in the methylotropic yeast Pichia pastoris. The PI-6 cDNA was modified to encode six histidine residues immediately after the initiation codon, and was placed under the control of the P. pastoris alcohol oxidase promoter in the vector pHIL-D2. On methanol induction, active recombinant PI-6 was produced within the yeast cells, and following cell lysis, was separated from yeast proteins by affinity chromatography using nickel nitrilo-tri-acetic acid (NTA) resin. The interaction of recombinant PI-6 with a range of serine proteinases was studied. Second order association rate constants ( k a) were derived for the interaction with trypsin (1.8 · 10 6 M −1s −1), thrombin (1.2 · 10 5 M −1 s −1), urokinase plasminogen activator (4.0 · 10 4 M −1s −1), plasmin (1.3 · 10 6 M −1s −1), and activated protein C (7.5 · 10 3 M −1 s −1). By monitoring complex formation, recombinant PI-6 was also shown to interact with factor Xa. No complex formation was observed with chymotrypsin, human leukocyte elastase, cathepsin G and tissue plasminogen activator, although PI-6 is apparently a substrate for chymotrypsin, leukocyte elastase and cathepsin G.