GENETIC ENGINEERING OF PROTEASE INHIBITORS Alpha1-ANTITRYPSIN AND HIRUDIN

Abstract

Site-directed mutagenesis was employed to express different variants of a,antitrypsin in a recombinant strain of Escherichia coli. The first set of variants was designed to render the inhibitor stable under oxidative conditions which reduce the activity of the natural molecule. This was achieved by replacing the methionine residue in position 358 (Pl position) by either valine or leucine. In vitro testing of the variant inhibitors under conditions which mimic the in vivo situation of oxidative stress in the epithelial lining fluid of the lung of cigarette smokers confirmed that they retained their inhibitory efficiency against neutrophil elastase. Furthermore site-directed mutagenesis was used to replace the methionine??® residue with an arginine in order to change the inhibitory specificity of a,antitrypsin from neutrophil elastase to a-thrombin. The design of this inhibitor was based upon the known specificity of a-thrombin for arginine and also upon the presence of an arginine residue in the Pl position of the natural a-thrombin inhibitor antithrombin III. Subsequent in vitro and in vivo evaluation of this variant confirmed that a potent inhibitor of a-thrombin had been designed. Based on further sequence homology studies with other members of the serine protease inhibitor (serpin) family another a@,antitrypsin variant with more pronounced inhibitory effects on plasma kallikrein and factor XIIa was designed. This was achieved by replacing the proline357 residue in the arginine358 variant with an alanine. The double variant matched the Cl-inhibitor in its P2 and P1 positions and proved to be more effective against plasma kallikrein and factor XIIa both in vitro and in vivo than the mutant with only an arginine?>® residue. In a separate set of experiments variants of the naturally occurring a-thrombin inhibitor hirudin were designed and expressed in a recombinant yeast strain. Site-directed mutagenesis experiments established the importance of having a basic residue such as lysine or arginine in position 47 of the inhibitor to obtain efficient thrombin inhibition. In addition it was shown that a replacement of lysine?5 with a threonine residue did not alter the inhibition efficiency thus indicating that the surface loop region around this residue is not involved in the interaction with a-thrombin.