Studies on human biliverdin-IX alpha reductase and linear tetrapyrrole signaling

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.