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Title: The cloning, expression and characterisation of PbrR from Pseudomonas putida KT2440
Author: Ansir, Nagwa Hassan
ISNI:       0000 0004 5363 8710
Awarding Body: Bangor University
Current Institution: Bangor University
Date of Award: 2014
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Highly selective lead-binding proteins (PbrR family) have been characterised in relatively few biological systems. This thesis reports the first research on cloning, expression and characterisation of native and mutant PbrR froin Pseudomonas putida KT2440 (PbrR(Pp)). Three cysteine residues are believed to play a vital role in ligand binding and discrimination (Cys77, Cys112, and Cys119). To investigate the role of these residues, cysteine was mutated to single or multiple mutants of either alanine, (C77A, C77AJCl12A, and C77AJC112/119A) or aspartic acid (C77D, C77D/Cl12D, and C77D/Cl12D1119 D). A recombinant DNA vector was generated by cloning the PbrR(Pp) gene into the pET28 (+) plasmid. Site directed mutagenesis was utilised to generate the desired mutants using the recombinant DNA plasmid as a template. The recombinant and all mutants were successfully expressed in the E. coli strain BL21 (DE3), as evidenced by DNA sequencing and SDS PAGE analysis, and purified using HiTrap affinity columns. UVNis spectroscopy, atomic absorption spectroscopy and protein concentration assays showed that lead was partially incorporated into the as-isolated PbrR protein from Pseudomonas putida KT2440 (PbrR(Pp)) with a protein:lead ratio of (1 :0.9) Reconstitution of the holoprotein, either via dialysis or titration, resulted in formation of a Pb-PbrR(Pp) complex, indicating that each PbrR(Pp) dimer binds one Pb2 + ion. A characteristic charge transfer band in the UV/Vis spectrum of Pb-PbrR(Pp) was observed at 332 nm (ε/max = 3,720 M-1 cm-1). For the mutant proteins, only the Cys77D (aspartic acid) mutant had a nearly 1:1 lead:protein with percentage lead saturation in Pb-PbrR-C77D was 88% after reconstitution with lead, and an associated blue shifted charge transfer band appeared at 318 nm (ε/max=3889 M-1 cm-1). The other mutants did not exhibit any clearly defined charge transfer band and the percentage lead saturation were range (15%--40%). Currently, 207Pb NMR spectroscopy has not been widely used by many research groups to study lead enzyme complexes. In this thesis, 207Pb NMR spectroscopy has been used to characterise a model system, a Pb-glutathione complex, and PbPbrR( Pp). Comparison of the observed chemical shifts (singlet centred at 2765 ppm) confirmed that the Pb-PbrR(Pp) has a Pb-S3 coordination geometry.
Supervisor: Murphy, Loretta Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available