An efficient method for expression in Escherichia coli and purification of the extracellular ligand binding domain of the human TGFβ type II receptor

Gasparian, Marine E.; Elistratov, P. A.; Yakimov, S. A.; Долгих, Д. А.; Kirpichnikov, Mikhaǐl P.

doi:10.1016/j.jbiotec.2010.04.013

Cited by 5 publications

(6 citation statements)

References 34 publications

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“…Several receptors of the superfamily, including BMPRIa, BMPRIb, BMPRII, and TβRII have been previously produced as soluble proteins in E. coli by expressing them as fusion proteins with thioredoxin [62,80–84]. The advantage of producing the receptors in this manner is that the proteins remain soluble and can be extracted from the bacteria in their native form.…”

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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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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“…Although a thioredoxin fusion enhanced the solubility of chicken Trx-TBRII-ECD [32,33], production of the corresponding human Trx-TBRII-ECD fusion resulted in soluble but nonfunctional protein that failed to bind TGF-β1. A protocol for folding Trx-TBRII-ECD on Ni–NTA agarose was previously reported [13]; however, it employs a high concentration of arginine at pH 8.0, which interferes with protein binding to Ni–NTA agarose [34]. Still, we were inspired by this heterogenous folding strategy [13] to examine on-plate folding as a means of rapidly identifying improved conditions.…”

Section: Resultsmentioning

confidence: 99%

“…A protocol for folding Trx-TBRII-ECD on Ni–NTA agarose was previously reported [13]; however, it employs a high concentration of arginine at pH 8.0, which interferes with protein binding to Ni–NTA agarose [34]. Still, we were inspired by this heterogenous folding strategy [13] to examine on-plate folding as a means of rapidly identifying improved conditions. For this assay, soluble Trx-TBRII-ECD was immobilized onto a Ni–NTA-coated 96-well plate (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Buffered solutions were screened for their ability to support folding (200 μL) by adding them to each well and holding the samples at 4 °C for 40 h. The plate was then washed with phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBST). The relative amount of correctly folded TBRII-ECD was measured directly on the plate using a previously described protocol that monitors TGF-β1 interaction with the receptor [13]. Briefly, the plate was blocked with 200 μL of 5% BSA in PBST at 37 °C for 1.5 h. TGF-β1 (5 ng/mL, 100 μL, Cell Signaling Technology) was added.…”

Section: Methodsmentioning

confidence: 99%

“…To pursue the required structural and biophysical studies, large quantities of TBRs are required. In our hands, the previously reported expression and purification protocols resulted in variable yields of functional TBRII-ECD [11-13]. The variability resulted from the folding step; therefore, we sought to develop an on-plate screen to optimize folding.…”

Section: Introductionmentioning

confidence: 99%

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The non-detergent sulfobetaine-201 acts as a pharmacological chaperone to promote folding and crystallization of the type II TGF-β receptor extracellular domain

Wangkanont

Forest

Kiessling

2015

Protein Expression and Purification

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The roles of the extracellular domain of type II TGF-β receptor (TBRII-ECD) in physiological processes ranging from development to cancer to wound healing render it an attractive target for exploration with chemical tools. For such applications, large amounts of active soluble protein are needed, but the yields of TBRII-ECD we obtained with current folding protocols were variable. To expedite the identification of alternative folding conditions, we developed an on-plate screen. This assay indicated that effective folding additives included the non-detergent sulfobetaine-201 (NDSB-201). Although NDSB-201 can facilitate protein folding, the mode by which it does so is poorly understood. We postulated that specific interactions between NDSB-201 and TBRII-ECD might be responsible. Analysis by X-ray crystallography indicates that the TBRII-ECD possesses a binding pocket for NDSB-201. The pyridinium group of the additive stacks with a phenylalanine side chain in the binding site. The ability of NDSB-201 to occupy a pocket on the protein provides a molecular mechanism for the additive’s ability to minimize TBRII-ECD aggregation and stabilize the folded state. NDSB-201 also accelerates TBRII-ECD crystallization, suggesting it may serve as a useful crystallization additive for proteins refolded with it. Our results also suggest there is a site on TBRII-ECD that could be targeted by small-molecule modulators.

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New Strategy for High-Level Expression and Purification of Biologically Active Monomeric TGF-β1/C77S in Escherichia coli

et al. 2014

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An efficient method for expression in Escherichia coli and purification of the extracellular ligand binding domain of the human TGFβ type II receptor

Cited by 5 publications

References 34 publications

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

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

The non-detergent sulfobetaine-201 acts as a pharmacological chaperone to promote folding and crystallization of the type II TGF-β receptor extracellular domain

New Strategy for High-Level Expression and Purification of Biologically Active Monomeric TGF-β1/C77S in Escherichia coli

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