The intracellular Ig fold : a robust protein scaffold for the engineering of molecular recognition

Bruning, Marc; Barsukov, Igor; Franke, Barbara; Barbieri, Sonia; Volk, Martin; Leopoldseder, Sonja; Ucurum, Zöhre; Mayans, Olga

The intracellular Ig fold : a robust protein scaffold for the engineering of molecular recognition

Dateien

Bruning_0-406407.pdfGröße: 374.34 KBDownloads: 208

Datum

2012

Autor:innen

Bruning, Marc

Barsukov, Igor

Franke, Barbara

Barbieri, Sonia

Volk, Martin

Leopoldseder, Sonja

Ucurum, Zöhre

Mayans, Olga

Open Access-Veröffentlichung

Open Access Green

Sammlungen

Biologie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

Protein Engineering Design and Selection. 2012, 25(5), pp. 205-212. ISSN 1741-0126. eISSN 1741-0134. Available under: doi: 10.1093/protein/gzs007

Zusammenfassung

Protein scaffolds that support molecular recognition have multiple applications in biotechnology. Thus, protein frames with robust structural cores but adaptable surface loops are in continued demand. Recently, notable progress has been made in the characterization of Ig domains of intracellular origin--in particular, modular components of the titin myofilament. These Ig belong to the I(intermediate)-type, are remarkably stable, highly soluble and undemanding to produce in the cytoplasm of Escherichia coli. Using the Z1 domain from titin as representative, we show that the I-Ig fold tolerates the drastic diversification of its CD loop, constituting an effective peptide display system. We examine the stability of CD-loop-grafted Z1-peptide chimeras using differential scanning fluorimetry, Fourier transform infrared spectroscopy and nuclear magnetic resonance and demonstrate that the introduction of bioreactive affinity binders in this position does not compromise the structural integrity of the domain. Further, the binding efficiency of the exogenous peptide sequences in Z1 is analyzed using pull-down assays and isothermal titration calorimetry. We show that an internally grafted, affinity FLAG tag is functional within the context of the fold, interacting with the anti-FLAG M2 antibody in solution and in affinity gel. Together, these data reveal the potential of the intracellular Ig scaffold for targeted functionalization.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Schlagwörter

differential scanning fluorimetry, infrared spectroscopy, intracellular Ig domain, NMR spectroscopy, protein scaffold engineering for peptide display

Zitieren

ISO 690

BRUNING, Marc, Igor BARSUKOV, Barbara FRANKE, Sonia BARBIERI, Martin VOLK, Sonja LEOPOLDSEDER, Zöhre UCURUM, Olga MAYANS, 2012. The intracellular Ig fold : a robust protein scaffold for the engineering of molecular recognition. In: Protein Engineering Design and Selection. 2012, 25(5), pp. 205-212. ISSN 1741-0126. eISSN 1741-0134. Available under: doi: 10.1093/protein/gzs007

BibTex

@article{Bruning2012-05-01intra-38669,
  year={2012},
  doi={10.1093/protein/gzs007},
  title={The intracellular Ig fold : a robust protein scaffold for the engineering of molecular recognition},
  number={5},
  volume={25},
  issn={1741-0126},
  journal={Protein Engineering Design and Selection},
  pages={205--212},
  author={Bruning, Marc and Barsukov, Igor and Franke, Barbara and Barbieri, Sonia and Volk, Martin and Leopoldseder, Sonja and Ucurum, Zöhre and Mayans, Olga}
}

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