TGF-beta is a pleiotropic, multifunctional cytokine with potent immunoregulatory properties. Nearly all cells of leukocyte lineage express the cytokine. Although not completely understood, the role of TGF-beta has been established as the primary inhibitory cytokine in many processes including inflammatory responses, carcinogenesis and oral tolerance. TGF-beta treatment leads to cell-cycle arrest at the late G1 phase through the expression of a cyclin-dependent kinase inhibitor p27 molecule. The TGF-beta receptor consists of two chains, type I and II; both are receptor serine/threonine kinases. The binding of TGF-beta to the type II receptor recruits and activates the type I receptor, which in turn activates the SMAD signaling pathway, leading to the regulation of gene expression. Due to the lack of structural work, the ligand-binding region of both the receptor and TGF-beta remains unclear. Our objective is to determine the crystal structures of the TGF- beta receptor alone and in complex with TGF-beta. We hope to gain functional insight into the receptor activation. Our first attempt was to crystallize a baculovirus expressed human type II TGF-beta receptor (TBRII) in complex with TGF- beta1. Due to extensive glycosylation on the receptor, the crystallization trials with this receptor did not yield any crystals. Subsequent deglycosylation procedures yielded a partially deglycosylated receptor that produced small crystals when complexed with TGF-beta1. However, these crystals diffracted poorly in the X-ray beam and were not suitable for structure determination. To overcome the carbohydrate heterogeneity and low expression yield of the baculovirus expression system, we have attempted to express the soluble type II receptor in several bacteria expression vector systems. Due to a large number of disulfide bonds (12 cysteines) present in this receptor, previous attempts by other groups to reconstitute the bacteria expressed receptor have all failed. The first bacteria construct we made was a fusion protein of the type II TGF-beta receptor and GST. This resulted in the production of a functional receptor in the soluble fraction of cell lysates, as evident from a TGF- beta binding ELISA assays. However, the yield of TBRII from the GST fusion construct remained low. This was primarily due to the degradation of the linker region between GST and the receptor, that resulted in the majority eluted sample from a glutathione affinity column being free GST rather than the GST-TBRII fusion protein. A typical 10 liter bacteria expression experiment yielded less than 0.5 mg of purified receptor protein. To overcome the problem of low expression yield, we began to investigate the purification schemes by refolding methods, despite previous attempts by others not being successful. As a result, we subcloned the ecto-TBRII (without any fusion partner) into two Novagen's pET vectors, pET14 and pET30, both of which are driven by T7 polymerase. The expression of this construct resulted in large quantities of inclusion bodies. Subsequent refolding experiments yielded milligram quantities of TBRII that appears active in a TGF-beta ELISA assay. The crystallization experiments using this receptor have produced crystals. More recently, we have completed the crystal structure of this receptor at 1.1 angstrom resolution.
