Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.660212
Title: Biochemical and biophysical studies of FK506 binding proteins
Author: Opamawutthikul, Monluedee
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2005
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Abstract:
This thesis describes the expression, purification, and biochemical and biophysical characterization of a number of FK506 binding proteins. The main focus of the work is to help characterize the roles of played FKBP by thus providing a better understanding of their biological functions. Neurospora crassa FKBP22 – Expression, purification, crystallization, and partial structure determination of the 22 kDa FKBP protein were carried out. The enzymatic PPIase activity of the N-terminal FKBP domain was characterized with the kca/Km value of 4 x 106 M-1s-1. Sequence analysis showed high homology of this domain to the 12 kDa FKBP with the highly conserved peptidly-prolyl active site. The C-terminal sequence appeared to be unique among other FKBP sequences; being highly charged with a stretch of predicted amphipathic helical conformation. Dynamic light scattering studies suggest that the protein may also exist as a dimer in solution. Caenorhabiditis elegans FKBP48 – The only TPR protein in C. elegans were cloned. Three natural breakdown products of FKBP48 were purified and characterized: an intact full length, a δ30 fragment missing final 30 C-terminal residues, and a ST fragment missing the C-terminal TPR domain. Results of small angle X-ray scattering revealed similarity of domain architecture between FKBP48 and FKBP51. Caernorhabiditis elegans FKBP12, Caenorhabiditis elegans FKBP29, and Arabidopsis thaliana FKBP71 – FKBP12/29 were well expressed. The enzymatic PPIase activity of FKBP29 was characterized with the kca/Km value of 0.3 x 106 M-1s-1. However the protein yield was insufficient for further studies. Purification of FKBP12 was difficult to manage in this work. Protein aggregatin was noticed during cleavage of fusion maltose binding protein. The recombinant 71 kDa FKBP protein was expressed at low level. This could be due to its mild toxicity. In addition quality of the protein was poor with inherent instability.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.660212  DOI: Not available
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