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 (2012). The intracellular Ig fold: a robust protein scaffold for the engineering of molecular recognition. Protein engineering, design & selection, 25(5), pp. 205-12. Oxford: Oxford University Press 10.1093/protein/gzs007

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Ucurum Fotiadis, Zöhre

ISSN:

1741-0126

Publisher:

Oxford University Press

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 14:37

Last Modified:

17 Mar 2015 21:29

Publisher DOI:

10.1093/protein/gzs007

PubMed ID:

22355150

Web of Science ID:

000303341000001

URI:

https://boris.unibe.ch/id/eprint/15049 (FactScience: 222208)

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