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Covalent immobilization of recombinant fusion proteins with hAGT for single molecule force spectroscopy

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Abstract

A genetically modified form of the human DNA repair protein O6-alkylguanine-DNA-alkyltransferase (hAGT) was used to immobilize different recombinant hAGT fusion proteins covalently and selectively on gold and glass surfaces. Fusion proteins of hAGT with Glutathione S-Transferase and with tandem repeats of Titin Ig-domains, were produced and anchored via amino-polyethylene glycol benzylguanine. Anchoring was characterized and quantified with surface plasmon resonance, atomic force microscope and fluorescence measurements. Individual fusion proteins were unfolded by single molecule force spectroscopy corroborating the selectivity of the covalent attachment.

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Abbreviations

hAGT:

O6-alkylguanine-DNA-alkyltransferase

GST:

Glutathione S-Transferase

PEG:

Polyethylene glycol

BG:

Benzylguanine

SPR:

Surface plasmon resonance

AFM:

Atomic force microscope

EDC:

1-ethyl-3-(3-diaminopropyl)carbodiimide hydrochloride

NHS:

N-hydroxysuccinimide

GST:

Glutathione S-transferase

CMC:

Carboxymethylcellulose

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Acknowledgements

We thank Martin Benoit for advise and help. Special thank also to Jan Barnikow and Kai Johnsson from Covalys Switzerland who provided the BG-anchor and the hAGT vector. This work was supported by the DFG.

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Authors and Affiliations

  1. Lehrstuhl für Angewandte Physik, Sektion Physik, Ludwig-Maximilians-Universität München and Center for NanoScience, Amalienstrasse 54, 80799, Munich, Germany

    Stefan K. Kufer, Christian Albrecht, Kerstin Blank, Angelika Kardinal & Hermann E. Gaub

  2. Physik-Department E22, Technische Universität München, James-Franck-Strasse, 85747, Garching, Germany

    Hendrik Dietz & Matthias Rief

Authors
  1. Stefan K. Kufer
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  2. Hendrik Dietz
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  3. Christian Albrecht
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  4. Kerstin Blank
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  5. Angelika Kardinal
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  6. Matthias Rief
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  7. Hermann E. Gaub
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Corresponding author

Correspondence to Hermann E. Gaub.

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Kufer, S.K., Dietz, H., Albrecht, C. et al. Covalent immobilization of recombinant fusion proteins with hAGT for single molecule force spectroscopy. Eur Biophys J 35, 72–78 (2005). https://doi.org/10.1007/s00249-005-0010-1

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  • DOI: https://doi.org/10.1007/s00249-005-0010-1

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