Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596753
Title: Recombinant TGF-beta type I and type II receptor domains
Author: Bolton, L. W. Y. T.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1999
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Abstract:
Transforming growth factor-β (TGF-β) is a multifunctional cytokine that signals by binding to two related transmembrane serine/threonine kinases called the type I and type II receptors. The in vitro binding properties of these receptors for the TGF-β1 and TGF-β3 isoforms were investigated by developing expression systems for recombinant extracellular domains (ECDs) of the type I and type II receptors, which also enabled the interaction of the two receptor domains to be examined. 1. A variety of vectors were constructed incorporating the fusion protein glutathione S transferase (GST) and peptide tags to promote purification of the ECDs from bacterial lysates (E.coli TG1 and BL21 (DE3) strains). 2. The optimum conditions for purification and yield of the recombinant proteins from lysed bacteria were examined for vector and bacterial systems for both type I and type II ECDs. 3. The ECD of the type I receptor was found to bind TGF-β1 in vitro, in the absence of the type II receptor. Binding was retained in an ECD cleaved from the GST domain and therefore did not depend on the presence of the fusion protein or other peptide tags. 4. The ECD of the type II receptor bound both TGF-β1 and TGF-β3 isoforms, consistent with previous in vitro data, whereas the type I receptor ECD did not bind TGF-β3. 5. Attempts to develop an ELISA for TGF-β1 using the type I and II receptor ECDs as capture and detection agents revealed that the two receptor ECDs associate with each other in the absence of TGF-β1. 6. A mammalian cell expression system to produce TGF-β3 was developed to enable biochemical and structural interactions of TGF-β3.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.596753  DOI: Not available
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