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Monitoring the assembly of antibody-binding membrane protein arrays using polarised neutron reflection

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Abstract

Protein arrays are used in a wide range of applications. The array described here binds IgG antibodies, produced in rabbit, to gold surfaces via a scaffold protein. The scaffold protein is a fusion of the monomeric E. coli porin outer membrane protein A (OmpA) and the Z domain of Staphylococcus aureus protein A. The OmpA binds to gold surfaces via a cysteine residue in a periplasmic turn and the Z domain binds immunoglobulins via their constant region. Polarised Neutron Reflection is used to probe the structure perpendicular to the gold surface at each stage of the assembly of the arrays. Polarised neutrons are used as this provides a means of achieving extra contrast in samples having a magnetic metal layer under the gold surface. This contrast is attained without resorting to hydrogen/deuterium exchange in the biological layer. Polarised Neutron Reflection allows for the modelling of many and complex layers with good fits. The total thickness of the biological layer immobilised on the gold surface is found to be 187 Å and the layer can thus far be separated into its lipid, protein and solvent parts.

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Abbreviations

BME:

2-Mercaptoethanol

DTT:

Dithiothreitol

IgG:

Immunoglobulin G

IMAC:

Immobilized metal affinity chromatography

IPTG:

Isopropyl-β-d-thiogalactopyranoside

OG:

n-Octylglucoside

OmpA:

Outer membrane protein A

OmpAZ:

Circularly permuted OmpA with two SpA Z domains fused at the N terminus

PNR:

Polarised Neutron Reflection

Q:

Momentum transfer

SDS:

Sodium dodecyl sulphate

SLD:

Scattering length density

SpA:

Staphylococcus aureus protein A

TCEP:

Tris(2-carboxy-ethyl)phosphine hydrochloride

thioPEG:

Polyethylene glycol with a thiolalkane functional group

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Acknowledgments

The authors would like to thank Frank Heinrich for help with preparation of the gold surfaces. The Biotechnology and Biological Sciences Research Council, the Science and Technology Facilities Council and Orla Protein Technologies Ltd funded this work.

The identification of any commercial product or trade name does not imply endorsement or recommendation by the National Institute of Standards and Technology.

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Authors and Affiliations

  1. Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK

    Anton P. Le Brun & Jeremy H. Lakey

  2. ISIS, STFC Rutherford Appleton Laboratory, Didcot, Oxfordshire, OX11 0QX, UK

    Stephen A. Holt

  3. Orla Protein Technologies Ltd, Nanotechnology Centre, Herschel Building, Newcastle upon Tyne, NE1 7RU, UK

    Deepan S. Shah

  4. NIST Centre for Neutron Research, Gaithersburg, MD, 20899, USA

    Charles F. Majkrzak

Authors
  1. Anton P. Le Brun
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  2. Stephen A. Holt
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  3. Deepan S. Shah
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  4. Charles F. Majkrzak
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  5. Jeremy H. Lakey
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Corresponding author

Correspondence to Jeremy H. Lakey.

Additional information

Advanced neutron scattering and complementary techniques to study biological systems. Contributions from the meetings, ‘‘Neutrons in Biology’’, STFC Rutherford Appleton Laboratory, Didcot, UK, 11–13 July and ‘‘Proteins At Work 2007’’, Perugia, Italy, 28–30 May 2007.

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Le Brun, A.P., Holt, S.A., Shah, D.S. et al. Monitoring the assembly of antibody-binding membrane protein arrays using polarised neutron reflection. Eur Biophys J 37, 639–645 (2008). https://doi.org/10.1007/s00249-008-0291-2

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  • DOI: https://doi.org/10.1007/s00249-008-0291-2

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