EXPRESSION OF HUMAN AMYLOID PRECURSOR PROTEIN ECTODOMAINS IN PICHIA-PASTORIS - ANALYSIS OF CULTURE CONDITIONS, PURIFICATION, AND CHARACTERIZATION

We have examined the use of the yeast Pichia pastoris for expression o f the human amyloid precursor protein (APP). The ectodomains of the is oforms APP695, APP751, and APP770 were expressed in both P. pastoris p rotease-deficient strain SMD1163 and wild-type strain GS115, using the secretion vector pHIL-S1. Expression of recombinant APP in each of th ese strains produced intact recombinant protein, together with a small number of breakdown products. The levels of these breakdown products were not significantly altered by expression in the protease-deficient strain compared with wild-type GS115. The effects of induction time a nd medium composition on recombinant APP stability were also examined, After optimization of expression and culture conditions, baffled shak er flask cultures of clones selected for high expression routinely yie lded 13-24 mg/liter recombinant protein following a two-step purificat ion procedure. The recombinant isoforms possessed the heparin binding, metal binding, and Kunitz-type protease inhibitor properties of human brain-derived APP. These data indicate that P. pastoris is an appropr iate laboratory-scale expression system for production of sufficient q uantities of recombinant APP for use in biological studies. (C) 1997 A cademic Press.