Studies on human biliverdin-IX alpha reductase and linear tetrapyrrole signaling
Citation:
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 259Download Item:
Dunne TCD THESIS 6353 Studies on.pdf (PDF) 149.8Mb
Abstract:
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.
Author: Dunne, Aisling
Advisor:
Mantle, TimQualification name:
Doctor of Philosophy (Ph.D.)Publisher:
Trinity College (Dublin, Ireland). School of Biochemistry and ImmunologyNote:
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Biochemistry, Ph.D., Ph.D. Trinity College DublinLicences: