Recombinant production, purification, crystallization, and structure analysis of human transforming growth factor β2 in a new conformation
| Autor: | Laura del Amo-Maestro, Laura Marino-Puertas, Theodoros Goulas, F. Xavier Gomis-Rüth |
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| Přispěvatelé: | Generalitat de Catalunya, Ministerio de Economía y Competitividad (España), Fundació La Marató de TV3, Ministerio de Ciencia, Innovación y Universidades (España) |
| Rok vydání: | 2019 |
| Předmět: |
Models
Molecular Protein Conformation alpha-Helical Multidisciplinary Expression systems Recombinant Fusion Proteins lcsh:R Gene Expression lcsh:Medicine Crystallography X-Ray Article Tissue Culture Techniques Transforming Growth Factor beta2 HEK293 Cells Protein Domains Humans Protein Isoforms Histidine Protein Conformation beta-Strand lcsh:Q Crystallization lcsh:Science Structural biology Oligopeptides Plasmids |
| Zdroj: | Digital.CSIC. Repositorio Institucional del CSIC instname Scientific Reports, Vol 9, Iss 1, Pp 1-11 (2019) Scientific Reports |
| ISSN: | 2015-6448 |
| Popis: | © The Author(s) 2019. Transforming growth factor β is a disulfide-linked dimeric cytokine that occurs in three highly related isoforms (TGFβ1–TGFβ3) engaged in signaling functions through binding of cognate TGFβ receptors. To regulate this pathway, the cytokines are biosynthesized as inactive pro-TGFβs with an N-terminal latency-associated protein preceding the mature moieties. Due to their pleiotropic implications in physiology and pathology, TGFβs are privileged objects of in vitro studies. However, such studies have long been limited by the lack of efficient human recombinant expression systems of native, glycosylated, and homogenous proteins. Here, we developed pro-TGFβ2 production systems based on human Expi293F cells, which yielded >2 mg of pure histidine- or Strep-tagged protein per liter of cell culture. We assayed this material biophysically and in crystallization assays and obtained a different crystal form of mature TGFβ2, which adopted a conformation deviating from previous structures, with a distinct dimeric conformation that would require significant rearrangement for binding of TGFβ receptors. This new conformation may be reversibly adopted by a certain fraction of the mature TGβ2 population and represent a hitherto undescribed additional level of activity regulation of the mature growth factor once the latency-associated protein has been separated. This study was supported in part by grants from Spanish and Catalan public and private bodies (grant/fellowship references BFU2015-64487R; MDM-2014-0435; JCI-2012-13573; BES-2015-074583; BES-2013-064651; 2017SGR3; and Fundació “La Marató de TV3” 201815). Te Structural Biology Unit of IBMB is a “María de Maeztu” Unit of Excellence from the Spanish Ministry of Science, Innovation and Universities. |
| Databáze: | OpenAIRE |
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