A novel TWO-STEP renaturation procedure for efficient production of recombinant BMP-2

Einem, Sabrina von; Schwarz, Elisabeth; Rudolph, Rainer

doi:10.1016/j.pep.2010.03.009

Cited by 37 publications

(28 citation statements)

References 19 publications

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“…Purified Grem2, GDF5, BMP2, NBL1, and Follistatin were generated utilizing previously published protocols (Cash et al, 2009; Einem et al, 2010; Nolan et al, 2015; 2013). Grem2 mutants were generated using the quickchange mutagenesis and produced similar to wild-type Grem2.…”

Section: Methodsmentioning

confidence: 99%

Structure of Gremlin-2 in Complex with GDF5 Gives Insight into DAN-Family-Mediated BMP Antagonism

et al. 2016

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Summary The DAN-family, including Gremlin-1 and Gremlin-2 (Grem1 and Grem2), represents a large family of secreted BMP antagonists. However, how DAN proteins specifically inhibit BMP signaling has remained elusive. Here, we report the structure of Grem2 bound to GDF5 at 2.9 Å resolution. The structure reveals two Grem2 dimers binding perpendicularly to each GDF5 monomer, resembling an H-like structure. Comparison to the unbound Grem2 structure reveals a dynamic N-terminus that undergoes significant transition upon complex formation, leading to simultaneous interaction with the type I/type II receptor motifs on GDF5. Binding studies show that DAN-family members can interact with BMP-type I receptor complexes, whereas Noggin outcompetes the type I receptor for ligand binding. Interestingly, Grem2-GDF5 forms a stable aggregate-like structure, in vitro, not clearly observed for other antagonists, including Noggin and Follistatin. These findings exemplify the structural and functional diversity across the various BMP antagonist families.

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Section: Methodsmentioning

confidence: 99%

Structure of Gremlin-2 in Complex with GDF5 Gives Insight into DAN-Family-Mediated BMP Antagonism

et al. 2016

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“…Thus, the protein concentration must be adjusted to achieve a balance between minimizing loss due to increased formation of aggregates at higher concentration, and a slow rate of dimer formation at low protein concentrations. Though not yet in widespread use, it has been proposed that the folding be performed in two phases, one in which native monomers are formed, and another is which the native monomers are oxidized into disulfide-linked dimers [50]. To demonstrate feasibility, BMP-2 was initially folded under relatively dilute conditions (0.2 mg/mL) in the presence of redox buffers that promote disulfide exchange (0.1 mM reduced glutathione, 0.1 mM oxidized glutathione), and after a significant fraction of protein had folded into native monomers, the protein was concentrated five-fold (1 mg/mL) and transferred into buffer that only included oxidized glutathione (25 mM).…”

Section: Methodsmentioning

confidence: 99%

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

Huang

Hinck²

2016

Methods in Molecular Biology

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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.

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“…Recombinant mature BMP2 was purified as previously described and diluted in sodium acetate buffer (pH 4.5) prior to immobilization on a CM5 sensor chip using the standard EDC/NHS primary amine coupling chemistry and according to the manufacturer’s protocol [25,26]. To determine the binding kinetics of Grem2 WT and mutants, serial dilutions in HBS–EP buffer [20 mM HEPES (pH 7.4), 150 mM NaCl, 3.4 mM EDTA, and 0.005% v/v P20 surfactant] were injected at a constant flow rate of 30 μl/min for an association of 3 min and allowed to dissociate for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Analysis and identification of the Grem2 heparin/heparan sulfate-binding motif

Kattamuri

Nolan

Thompson

2017

Biochemical Journal

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Bone morphogenetic proteins (BMPs) are regulated by extracellular antagonists of the DAN (differential screening-selected gene aberrative in neuroblastoma) family. Similar to the BMP ligands, certain DAN family members have been shown to interact with heparin and heparan sulfate (HS). Structural studies of DAN family members Gremlin-1 and Gremlin-2 (Grem2) have revealed a dimeric growth factor-like fold where a series of lysine residues cluster along one face of the protein. In the present study, we used mutagenesis, heparin-binding measurements, and cell surface-binding analysis to identify lysine residues that are important for heparin/HS binding in Grem2. We determined that residues involved in heparin/HS binding, while not necessary for BMP antagonism, merge with the heparin/HS-binding epitope of BMP2. Furthermore, the Grem2–BMP2 complex has higher affinity for heparin than the individual proteins and this affinity is not abrogated when the heparin/HS-binding epitope of Grem2 is attenuated. Overall, the present study shows that the Grem2 heparin/HS and BMP-binding epitopes are unique and independent, where, interestingly, the Grem2–BMP2 complex exhibits a significant increase in binding affinity toward heparin moieties that appear to be partially independent of the Grem2 heparin/HS-binding epitope.

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A novel TWO-STEP renaturation procedure for efficient production of recombinant BMP-2

Cited by 37 publications

References 19 publications

Structure of Gremlin-2 in Complex with GDF5 Gives Insight into DAN-Family-Mediated BMP Antagonism

Structure of Gremlin-2 in Complex with GDF5 Gives Insight into DAN-Family-Mediated BMP Antagonism

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

Analysis and identification of the Grem2 heparin/heparan sulfate-binding motif

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