Production, Isolation, and Structural Analysis of Ligands and Receptors of the TGF-β Superfamily

Abstract

The ability to understand the molecular mechanisms by which secreted signaling proteins of the TGF-β superfamily assemble their cell surface receptors into complexes to initiate downstream signaling is dependent upon the ability to determine atomic-resolution structures of the signaling proteins, the ectodomains of the receptors, and the complexes that they form. The structures determined to date have revealed major differences in the overall architecture of the signaling complexes formed by the TGF-βs and BMPs, which has provided insights as to how they have evolved to fulfill their distinct functions. Such studies, have however, only been applied to a few members of the TGF-β superfamily, which is largely due to the difficulty of obtaining milligram-scale quantities of highly homogenous preparations of the disulfide-rich signaling proteins and receptor ectodomains of the superfamily. Here we describe methods used to produce signaling proteins and receptor ectodomains of the TGF-β superfamily using bacterial and mammalian expression systems and procedures to purify them to homogeneity.

An engineered transforming growth factor β (TGF-β) monomer that functions as a dominant negative to block TGF-β signaling

“…The folding of the mini-monomers differed greatly; a large portion of the mmTGF-␤2 remained soluble during the folding and yielded large amounts of monomeric protein after purification by cation exchange chromatography, whereas only a small amount of mmTGF-␤1 and mmTGF-␤3 remained soluble during the folding, and either no monomeric protein (TGF-␤1) or a very small amount of monomeric protein (TGF-␤3) was obtained after purification by cation exchange chromatography. This pattern mirrors the pattern previously observed for the folding of TGF-␤ homodimers from full-length wild type monomers (38) and likely reflects differences in the intrinsic propensity of the monomers to properly form the four intramolecular disulfides characteristic of each monomer. mmTGF-␤2 was the least desired variant, due to the expected low affinity for binding T␤RII.…”

“…The cell line used to produce TGF-␤1, and the accompanying procedure to isolate the mature disulfide-linked TGF-␤1 homodimer from the conditioned medium, has been described previously (46) and was kindly provided from Dr. Peter Sun (NIAID, National Institutes of Health, Rockville, MD). Mouse homodimeric TGF-␤2 (TGF-␤2), human homodimeric TGF-␤3 (TGF-␤3), and variants, including homodimeric N-terminal avi-tagged (47) TGF-␤3 (avi-TGF-␤3), monomeric TGF-␤2 (mTGF-␤2), monomeric TGF-␤3 (mTGF-␤3), mini-monomeric TGF-␤1 (mmTGF-␤1), mini-monomeric TGF-␤2 (mmTGF-␤2), mini-monomeric TGF-␤3 (mmTGF-␤3), mini-monomeric TGF-␤2 with seven substitutions to enable high affinity T␤RII binding (mmTGF-␤2-7M), and mini-monomeric N-terminal avi-tagged (47) TGF-␤2 with seven substitutions to enable high affinity T␤RII binding (avi-mmTGF-␤2-7M), were expressed in E. coli, refolded from inclusion bodies into native folded disulfidelinked homodimers (TGF-␤2, TGF-␤3, avi-TGF-␤3) or monomers (mTGF-␤1, mTGF-␤2, mTGF-␤3, mmTGF-␤1, mmTGF-␤2, mmTGF-␤3, mmTGF-␤2-7M, avi-mmTGF-␤2-7M), and purified to homogeneity using high resolution cation exchange chromatography (Source Q, GE Healthcare) as described previously (38). The nomenclature and features of the dimeric and monomeric TGF-␤s used in this study are summarized in the supplemental Table S1, and the complete sequences are shown in supplemental Fig.…”

“…The inclusion bodies were isolated, and after reconstitution and purification in denaturant, the mini-monomers were renatured by dilution into CHAPScontaining buffer at pH 9.0 as described previously (38). The folding of the mini-monomers differed greatly; a large portion of the mmTGF-␤2 remained soluble during the folding and yielded large amounts of monomeric protein after purification by cation exchange chromatography, whereas only a small amount of mmTGF-␤1 and mmTGF-␤3 remained soluble during the folding, and either no monomeric protein (TGF-␤1) or a very small amount of monomeric protein (TGF-␤3) was obtained after purification by cation exchange chromatography.…”

“…Biotinylated TGF-␤3 or mmTGF-␤2-7M was generated by expressing TGF-␤3 or mmTGF-␤2-7M with an N-terminal 15-amino acid avitag (47). avi-TGF-␤3 or avi-mmTGF-␤2-7M was bound to T␤RII in 10 mM Bicine, pH 8.0, and biotinylated by incubating with a catalytic amount of bacterially expressed BirA recombinase, biotin, and ATP at 37°C for 2 h as described (38). Biotinylated avitagged TGF-␤3 or avi-tagged TGF-␤2-7M was bound to a C4 reverse phase column equilibrated with 94.9% water, 5% acetonitrile, 0.1% trifluoroacetic acid and eluted with a linear acetonitrile gradient.…”

Binding Properties of the Transforming Growth Factor-β Coreceptor Betaglycan: Proposed Mechanism for Potentiation of Receptor Complex Assembly and Signaling

“…Recombinant human TGF-β2 and the TGF-β2TM variant bearing Lys25 → Arg, Ile92 → Val, and Lys94 → Arg substitutions 9 were expressed in Escherichia coli as insoluble inclusion bodies and refolded and purified as described previously. 25 TβRI-ED and TβRII-ED were expressed in E. coli as insoluble inclusion bodies and refolded and purified as described previously. 26 , 27 …”