THE GOLGI SIALOGLYCOPROTEIN MG160, EXPRESSED IN PICHIA-PASTORIS, DOESNOT REQUIRE COMPLEX CARBOHYDRATES AND SIALIC-ACID FOR SECRETION AND BASIC FIBROBLAST GROWTH-FACTOR BINDING

MG160, a type I membrane sialoglycoprotein of the medial cisternae of the rat Golgi apparatus, shows high homology (over 90%) with CFR, a fi broblast growth factor receptor, and ESL-1, an E-selectin ligand of th e cell surface of murine myeloid cells. When Chinese Hamster Ovary (CH O) cells were stably transfected with a cDNA lacking the transmembrane and C-terminus cytoplasmic domain of MG160 (Delta TMCT), a fully proc essed protein of 160 kDa apparent molecular mass was recovered in the culture medium. When these cells were treated with tunicymycin, a 130- to 140-kDa protein was immunoprecipitated from the culture medium. A construct lacking the signal sequence, the single transmembrane, and t he cytoplasmic domains of MG160 (Delta TMCT-) was integrated at the HI S Pichia pastoris genome site using the expression vector pPIC 9 which possesses a yeast compatible signal sequence (Invitrogen). Recombinan t protein accumulated in the medium to approximately 10 mg/L. The yeas t recombinant protein lacked complex carbohydrates and sialic acid but bound I-125 bFGF. Similarly, rat MG160 subjected to deglycosylation b y peptide:N-glycosidase F (PNGase) bound I-125 bFGF. (C) 1997 Academic Press.