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Studies on human biliverdin-IX alpha reductase and linear tetrapyrrole signaling
Dunne, Aisling
THESIS 6353 Human Biliverdin-IXa reductase (BVR-A) has been cloned and overexpressed in E.coli as a GST- and Hexahistidine fusion protein. The full length cDNA encoding the enzyme has been amplified via PCR and hgated into the pGEX-KG and pTrcHis B expression vectors in order to produce the respective fusions. Induction of TGI cells transformed with the pGEX-BVR-A and pTrc-BVR-A constructs has culminated in the expression of a recombinant protein of the correct size and antigenicity in both cases. Purification of the GST-fusion protein on a glutathione affinity resin yields approximately 40 mg of fusion protein per litre of culture. The fusion protein has a molecular weight of 66 kDa, however, it is possible to remove the GST tag using the proteolytic enzyme, thrombin. The purified hBVR-A protein migrates on SDS-PAGE with a mobility corresponding to 40 kDa, however, mass spectroscopic analysis has confirmed the true relative molecular mass to be 34 kDa. A selenomethionine derivative of the recombinant hBVR-A protein has been prepared for use in Multiwavelength Anomalous Diffraction experiments and crystals diffracting at 3A have recently been obtained.
Keyword(s): Biochemistry, Ph.D.; Ph.D. Trinity College Dublin
Publication Date:
Type: Doctoral thesis
Peer-Reviewed: Unknown
Language(s): English
Institution: Trinity College Dublin
Citation(s): Aisling Dunne, 'Studies on human biliverdin-IX alpha reductase and linear tetrapyrrole signaling', [thesis], Trinity College (Dublin, Ireland). School of Biochemistry and Immunology, 2001, pp 259
Publisher(s): Trinity College (Dublin, Ireland). School of Biochemistry and Immunology
Supervisor(s): Mantle, Tim
First Indexed: 2018-12-08 06:20:37 Last Updated: 2018-12-08 06:20:37