The hsp150 delta-carrier confers secretion competence to the rat nerve growth factor receptor ectodomain in Saccharomyces cerevisiae.
When the extracellular domain of rat low-affinity nerve growth factor receptor (NGFRe) was synthesized in Saccharomyces cerevisiae with the signal peptide of invertase, NGFRe was translocated to the endoplasmic reticulum (ER) and retained there. However, when NGFRe was fused to the C-terminus of the hsp150 delta-carrier, the hsp150 delta-NGFRe fusion protein was efficiently secreted to the growth medium with no apparent retention in the ER. The NGFRe portion was disulphide-bonded and its single N-glycosylation site was occupied. The hsp150 delta-carrier is an N-terminal signal peptide-containing fragment of a yeast secretory glycoprotein. Hsp150 delta-NGFRe, harvested from the culture medium, inhibited the cross-linking of [125I]NGF to authentic NGFR on the surface of human melanoma cells. Moreover, [125I]NGF could be chemically cross-linked to secretory hsp150 delta-NGFRe, suggesting that the NGFRe portion had adopted a ligand-binding conformation. However, inhibition of the cross-linking by unlabelled NGF was less effective than in the case of the authentic receptor. The hsp150 delta-carrier may have potential in the production of mammalian proteins, which require elaborate folding and disulphide formation in the ER.
Simonen
M
M
Institute of Biotechnology, University of Helsinki, Finland.
Vihinen
H
H
Jämsä
E
E
Arumäe
U
U
Kalkkinen
N
N
Makarow
M
M
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D004220:Disulfides; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006023:Glycoproteins; D006026:Glycoside Hydrolases / Q000235:genetics; D006031:Glycosylation; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008545:Melanoma; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D051381:Rats; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014407:Tumor Cells, Cultured; D043324:beta-Fructofuranosidase
D004220:Disulfides; D006023:Glycoproteins; C074229:HSP150 protein, S cerevisiae; D006360:Heat-Shock Proteins; D009414:Nerve Growth Factors; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase