All individual domains of staphylococcal protein A show Fab binding |
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Authors: | Birger Janssona,Mathias Uhlé na,Per-Å ke Nygrena |
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Affiliation: | Department of Biochemistry and Biotechnology, Royal Institute of Technology, Teknikringen 30, S-100 44 Stockholm, Sweden |
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Abstract: | The interactions between the individual domains (E, D, A, B and C) of staphylococcal protein A (SPA) and Fc and Fab regions of human immunoglobulins were studied using real-time biospecific interaction analysis. An engineered domain Z, similar to fragment B but with a single glycine to alanine amino acid substitution, was also included in the study. The domains were expressed in Escherichia coli, affinity purified and immobilised onto sensor chip surfaces in a directed manner using a unique C-terminal cysteine residue engineered into the recombinant proteins. All domains bound to a recombinant human IgG1 Fc fragment with similar strength. For the first time, binding to human Fab was demonstrated for all native SPA domains, using both polyclonal F(ab′)2 and a recombinant scFv fragment as reagents. Interestingly, the engineered Z domain showed a considerably lower affinity for Fab as compared to the native domains. |
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Keywords: | Protein A Immunoglobulin Fab fragment Interaction Surface plasmon resonance |
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