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Self-Assembly of Protein Monolayers Engineered for Improved Monoclonal Immunoglobulin G Binding
Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK
Orla Protein Technologies Ltd, Biosciences Centre, International Centre for Life, Times Square, Newcastle upon Tyne, NE1 4EP, UK
ISIS Neutron Facility, STFC Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0QX, UK
Current address: Bragg Institute, Australian Nuclear Science and Technology Organisation, Locked Bag 2001, Kirrawee DC, NSW 2232, Australia.
* Author to whom correspondence should be addressed.
Received: 4 July 2011; in revised form: 4 July 2011 / Accepted: 10 August 2011 / Published: 15 August 2011
Abstract: Bacterial outer membrane proteins, along with a filling lipid molecule can be modified to form stable self-assembled monolayers on gold. The transmembrane domain of Escherichia coli outer membrane protein A has been engineered to create a scaffold protein to which functional motifs can be fused. In earlier work we described the assembly and structure of an antibody-binding array where the Z domain of Staphylococcus aureus protein A was fused to the scaffold protein. Whilst the binding of rabbit polyclonal immunoglobulin G (IgG) to the array is very strong, mouse monoclonal IgG dissociates from the array easily. This is a problem since many immunodiagnostic tests rely upon the use of mouse monoclonal antibodies. Here we describe a strategy to develop an antibody-binding array that will bind mouse monoclonal IgG with lowered dissociation from the array. A novel protein consisting of the scaffold protein fused to two pairs of Z domains separated by a long flexible linker was manufactured. Using surface plasmon resonance the self-assembly of the new protein on gold and the improved binding of mouse monoclonal IgG were demonstrated.
Keywords: membrane protein; gold; immunoglobulin; self-assembled monolayer; surface plasmon resonance
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Le Brun, A.P.; Shah, D.S.H.; Athey, D.; Holt, S.A.; Lakey, J.H. Self-Assembly of Protein Monolayers Engineered for Improved Monoclonal Immunoglobulin G Binding. Int. J. Mol. Sci. 2011, 12, 5157-5167.
Le Brun AP, Shah DSH, Athey D, Holt SA, Lakey JH. Self-Assembly of Protein Monolayers Engineered for Improved Monoclonal Immunoglobulin G Binding. International Journal of Molecular Sciences. 2011; 12(8):5157-5167.
Le Brun, Anton P.; Shah, Deepan S. H.; Athey, Dale; Holt, Stephen A.; Lakey, Jeremy H. 2011. "Self-Assembly of Protein Monolayers Engineered for Improved Monoclonal Immunoglobulin G Binding." Int. J. Mol. Sci. 12, no. 8: 5157-5167.