Simonen M, Vihinen H, Jämsä E, Arumäe U, Kalkkinen N, Makarow M
Institute of Biotechnology, University of Helsinki, Finland.
Yeast. 1996 Apr;12(5):457-66. doi: 10.1002/(SICI)1097-0061(199604)12:5%3C457::AID-YEA930%3E3.0.CO;2-D.
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.
当大鼠低亲和力神经生长因子受体（NGFRe）的细胞外结构域与转化酶信号肽一起在酿酒酵母中合成时，NGFRe被转运到内质网（ER）并保留在那里。然而，当NGFRe与hsp150δ载体的C末端融合时，hsp150δ-NGFRe融合蛋白被有效分泌到生长培养基中，在内质网中没有明显保留。NGFRe部分形成了二硫键，其单个N-糖基化位点被占据。hsp150δ载体是酵母分泌糖蛋白的含N末端信号肽的片段。从培养基中收获的hsp150δ-NGFRe抑制了[125I]NGF与人黑色素瘤细胞表面真实NGFR的交联。此外，[125I]NGF可以与分泌性hsp150δ-NGFRe化学交联，表明NGFRe部分采用了配体结合构象。然而，未标记的NGF对交联的抑制作用不如真实受体有效。hsp150δ载体在生产需要在内质网中进行精细折叠和二硫键形成的哺乳动物蛋白方面可能具有潜力。
