EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE KUNITZ-TYPE PROTEINASE-INHIBITOR DOMAIN OF THE AMYLOID BETA-PROTEIN PRECURSOR-LIKE PROTEIN-2

In this report we describe the use of the methylotrophic industrial ye ast Pichia pastoris as a host system for the large scale production of the Kunitz-type proteinase inhibitor (KPI) domain of the amyloid beta -protein precursor-like protein-2 (APLP-2). The expression plasmid for the KPI domain of APLP-2 encoded amino acids 305-364 of the APLP-2 cD NA (Slunt et al. (1994) J. Biol. Chem. 269, 2637-2644). The secreted 6 0 amino-acid product was purified to homogeneity and biochemically cha racterized. Amino-acid sequencing of the expressed KPI domain of APLP- 2 verified its integrity. The proteinase inhibitory properties of the KPI domain of APLP-2 were compared to those of the KPI domain of prote inase nexin-2/amyloid beta-protein precursor (PN-2/A beta PP). Both KP I domains potently inhibited trypsin and, to a lesser extent, chymotry psin, plasmin, and coagulation factors XIa and IXa. However, the KPI d omain of APLP-2 was a approximate to 20-fold less effective inhibitor of coagulation factor XIa compared to the KPI domain of PN-2/A beta PP . Similarly, the KPI domain of APLP-2 was a less effective anticoagula nt in coagulation based assays than the KPI domain of PN-2/A beta PP. These studies indicate that the KPI domains of PN-2/A beta PP and APLP -2 form a family of proteinase inhibitors although the former is a bet ter inhibitor of factor XIa and a more potent anticoagulant than the l atter.