High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid beta-protein precursor.
The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.
Wagner
S L
SL
Salk Institute Biotechnology/Industrial Associates, La Jolla, CA 92037-4641.
Siegel
R S
RS
Vedvick
T S
TS
Raschke
W C
WC
Van Nostrand
W E
WE
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D010957:Plasmids; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000235:genetics* / Q000494:pharmacology
C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D002352:Carrier Proteins; D058255:Protease Nexins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D014360:Trypsin Inhibitor, Kunitz Soybean; D010450:Endopeptidases