title: Expression of human amyloid precursor protein ectodomains in Pichia pastoris: analysis of culture conditions, purification, and characterization.
abstract: We have examined the use of the yeast Pichia pastoris for expression of the human amyloid precursor protein (APP). The ectodomains of the isoforms APP695, APP751, and APP770 were expressed in both P. pastoris protease-deficient strain SMD1163 and wild-type strain GS115, using the secretion vector pHIL-S1. Expression of recombinant APP in each of these 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 and medium composition on recombinant APP stability were also examined. After optimization of expression and culture conditions, baffled shaker flask cultures of clones selected for high expression routinely yielded 13-24 mg/liter recombinant protein following a two-step purification 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 appropriate laboratory-scale expression system for production of sufficient quantities of recombinant APP for use in biological studies.
authors.affiliation: Department of Pathology, University of Melbourne, Parkville, Victoria, Australia.
authors.forename: A
authors.initials: A
authors.lastname: Henry
authors.forename: C L
authors.initials: CL
authors.lastname: Masters
authors.forename: K
authors.initials: K
authors.lastname: Beyreuther
authors.forename: R
authors.initials: R
authors.lastname: Cappai
mesh_terms: D016564:Amyloid beta-Protein Precursor / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D003470:Culture Media / Q000494:pharmacology; D004355:Drug Stability; D010450:Endopeptidases / Q000009:adverse effects / Q000172:deficiency; D004789:Enzyme Activation; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000378:metabolism
chemical_list: D016564:Amyloid beta-Protein Precursor; D003470:Culture Media; D008670:Metals; D011994:Recombinant Proteins; D006493:Heparin; D014360:Trypsin Inhibitor, Kunitz Soybean; D010450:Endopeptidases