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Title: Characterisation of pre-equilibrium and equilibrium binding interactions between Peptostreptococcal protein L and human kappa light chains
Author: Housden, Nicholas George
ISNI:       0000 0001 3582 3312
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2001
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Site directed mutagenesis has been performed on a single Ig-binding domain of protein L from Peptostreptococcus magnus (PpL) in order to further our understanding of the nature of its interaction with human kappa light chain (κ-chain) and to produce constructs with modified binding properties. The interactions of each of the mutated PpL constructs with κ-chain have been characterised under equilibrium conditions using fluorescence spectroscopy, circular dichroism, isothermal titration calorimetry and enzyme linked immunosorbant assay. Site specific mutagenesis of Tyr53 or Leu57 has been shown to reduce the affinity of the equilibrium complex with κ-chain by 25 to 50-fold depending upon the substitution. Pre-equilibrium binding studies carried out by stopped-flow fluorescence spectroscopy have shown the mutated PpL constructs to maintain the two-phase binding process previously described for Tyr64→Trp PpL. The rate construct for the initial phase of binding is concentration dependent and corresponds to the formation of an encounter complex. This is followed by a second phase with a rate constant independent of concentration, corresponding to a conformational rearrangement to produce a high affinity complex. Affinity chromatography using immobilised Leu57 → His PpL as the affinity ligand allows the elution of IgG from immobilised PpL at a pH of 4.0, compared to the pH of 2.5 required for equivalent elution from commercially available immobilised 4 domain PpL.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry