Characterisation of phytochrome using monoclonal antibodies.
thesisposted on 19.11.2015, 09:11 by Mary Louise. Holdsworth
Characterisation of phytochrome using monoclonal antibodies Mary L. Holdsworth Native oat phytochrome has been purified to homogeneity and used to produce a panel of monoclonal antibodies (mAbs). Selection of mAbs followed early screening against native phytochrome by ELISA, and SDS-denatured phytochrome by "mini" western blotting. Six mAbs which recognised SDS-denatured phytochrome were mapped using proteolytically derived fragments of phytochrome and subsequent immunoblotting. LAS 31 and 33 map to the 6 kDa NH2-terminus and LAS 35 and 41 map to the adjacent 4 kDa sub-NH2- terminal domain. LAS 11 maps to the 64 kDa- chromophore-bearing domain and LAS 32 maps to between 74 and 88 kDa from the NH2-terminus on the COOH- terminal-half of the molecule. A novel protocol for the mapping of conformation-specific mAbs has been developed and used to assign LAS 21, 34 and 42 to the 64 kDa-chromophore-bearing domain. Determination of differential affinities towards Pr and Pfr demonstrated that LAS 42 exhibited a higher affinity for Pfr, LAS 31, 33, 34 and 35 exhibited a higher affinity for Pr. LAS 41 discriminates absolutely against Pfr. LAS 41 has therefore facilitated:- (i) the purification of PfrP, i.e. Pfr which is free of contaminating Pr, (ii) the development of an ELISA for phytochrome photoequilibrium, (iii) the first direct experimental evidence that phytochrome can exist as a stable heterodimer in vitro and (iv) an appraisal of ELISA protocols for determining differential affinities of mAbs towards Pr and Pfr. Spectral analyses of phytochrome in the presence of mAbs have underlined the importance of the 6 kDa NH2-terminus in the maintenance of the spectral integrity of the molecule but have also indicated that the 4 kDa sub-NH2-terminal domain also interacts with the chromophore. Cross reactivity studies amongst phytochrome from monocots and dicots demonstrate that the epitopes recognised by LAS 11, 31, 33, 35 and 41 are not highly conserved. However, LAS 32 recognises phytochrome from every plant species tested, and is therefore recognising a highly conserved region on the molecule.