Goetschy J F, Letourneur O, Cerletti N, Horisberger M A
Core Drug Discovery Technology, Pharmaceutical Research Department, Ciba-Geigy Ltd, Basel, Switzerland.
Eur J Biochem. 1996 Oct 15;241(2):355-62. doi: 10.1111/j.1432-1033.1996.00355.x.
The activation of the human transforming growth factor (TGF-beta) system begins with the cytokine-induced association of the extracellular domains of two structurally related receptor subunits. To study the protein-protein interactions between TGF-beta and the ligand-specific receptor subunit, the extracellular domain of the human TGF-beta receptor type II (T beta R-II) has been expressed as an intracellular protein in insect cells using the baculovirus expression system. The cDNA construct was engineered to encode amino acids 24-159 (the signal sequence 1-23 was lacking) preceded by one initiator methionine residue and six histidine residues added at the carboxy terminus. The soluble receptor accumulated in the cytoplasm of infected cells and was purified by one-step nickel-chelate affinity chromatography. The purified protein was not glycosylated; it migrated as a single band of apparent mass 19.5 kDa in SDS/polyacrylamide gels, and had a homogeneous N-terminal sequence. We have established a solid-phase binding assay using radioiodinated TGF-beta 3 and capture antibodies to immobilize the soluble receptor. In this assay, the apparent dissociation constant of the TGF-beta type-II receptor ectodomain for TGF-beta 3 was approximately 150 nM (this value is approximately 1000-fold higher than that of the cell-membrane receptor complex of living cells). The affinity of TGF-beta 3 for the unglycosylated ectodomain of T beta R-II from insect cells was lower than the affinity for the recombinant glycosylated ectodomain T beta R-II from mouse cells. The novel assay has been used to characterize affinities and specificities of TGF-beta 3, TGF-beta 2, corresponding mutants and hybrid proteins, as well as a related protein, BMP-2. The assay could also be used to search for inhibitors.
人类转化生长因子（TGF-β）系统的激活始于细胞因子诱导的两个结构相关受体亚基细胞外结构域的结合。为了研究TGF-β与配体特异性受体亚基之间的蛋白质-蛋白质相互作用，人类II型TGF-β受体（TβR-II）的细胞外结构域已利用杆状病毒表达系统在昆虫细胞中作为细胞内蛋白表达。构建的cDNA经设计可编码氨基酸24 - 159（缺少信号序列1 - 23），在其前面有一个起始甲硫氨酸残基，并在羧基末端添加了六个组氨酸残基。可溶性受体在受感染细胞的细胞质中积累，并通过一步镍螯合亲和层析进行纯化。纯化的蛋白未进行糖基化修饰；在SDS/聚丙烯酰胺凝胶中它以一条表观分子量为19.5 kDa的单带形式迁移，并且具有一致的N端序列。我们利用放射性碘化的TGF-β3和捕获抗体建立了一种固相结合测定法来固定可溶性受体。在该测定法中，TGF-βII型受体胞外域对TGF-β3的表观解离常数约为150 nM（该值比活细胞的细胞膜受体复合物的解离常数高约1000倍）。TGF-β3对昆虫细胞来源的未糖基化TβR-II胞外域的亲和力低于对小鼠细胞来源的重组糖基化TβR-II胞外域的亲和力。这种新的测定法已用于表征TGF-β3、TGF-β2、相应突变体和杂合蛋白以及相关蛋白BMP-2的亲和力和特异性。该测定法还可用于寻找抑制剂。
