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 yeast 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 cDNA (Slunt et al. (1994) J. Biol. Chem. 269, 2637-2644). The secreted 60 amino-acid product was purified to homogeneity and biochemically characterized. 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 proteinase nexin-2/amyloid beta-protein precursor (PN-2/A beta PP). Both KPI domains potently inhibited trypsin and, to a lesser extent, chymotrypsin, plasmin, and coagulation factors XIa and IXa. However, the KPI domain of APLP-2 was a approximately 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 anticoagulant 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 better inhibitor of factor XIa and a more potent anticoagulant than the latter.
Van Nostrand
W E
WE
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717-4025.
Schmaier
A H
AH
Neiditch
B R
BR
Siegel
R S
RS
Raschke
W C
WC
Sisodia
S S
SS
Wagner
S L
SL
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry*; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry*; D001483:Base Sequence; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
C489697:APLP1 protein, human; C084104:APLP2 protein, human; D016564:Amyloid beta-Protein Precursor; D000925:Anticoagulants; C078201:Aplp1 protein, mouse; C492952:Aplp2 protein, mouse; D009419:Nerve Tissue Proteins; D014360:Trypsin Inhibitor, Kunitz Soybean