Stability and binding studies on Ig-binding domains of Protein L from Peptostreptococcus magnus

Fenwick, Samuel Ben (2006) Stability and binding studies on Ig-binding domains of Protein L from Peptostreptococcus magnus. University of Southampton, Doctoral Thesis.

Abstract

Protein L is a 76-106 kDa multi-domain protein expressed on the surface of ~ 10% of Peptostreptococcus magnus strains.  Four to five of the domain are capable of binding Ig, via interaction with κ-chain.  Single (PpL) and double domains from Protein L of strain 3316 have been previously cloned into the AP10-2 and pQE-30 vectors, respectively and the proteins have been expressed and purified from E. coli.

The commercial purification of antibodies is a large market, and for PpL to be viable in this application, it must be resistant to the removal of contaminants from chromatographic media. This involves prolonged exposure to 0.5 M NaOH.  The most understand cause of protein degradation under alkali conditions is that of Asn deamidation.  In this study, the stability of Wt PpL and a range of Asn to Ala PpL mutants have been investigated under these conditions by circular dichroism, affinity chromatography and SDS-PAGE.  It was discovered that the N61A, N73A and Y64W mutations together dramatically increase the alkali stability of PpL by 11.4 fold.

Stopped flow experiments have been performed and suggest that the N61A, N73A and Y64W mutations do not affect the affinity of either of the two sites present on PpL for binding κ-chain. ELISA experiments indicate that the global affinity for human IgG is essentially unaffected. Gdn HC1 denaturation experiments have shown that these mutations do not affect the conformational stability of PpL.

Double domain PpL mutants have been extensively characterised by fluorescence, ITC, immunoprecipitation, NMR and circular dichroism experiments.

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