Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection
Marine Goffinet,Patrick Chinestra,Isabelle Lajoie-Mazenc,Claire Medale-Giamarchi,Gilles Favre,Jean-Charles Faye +5 more
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TLDR
The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development.Abstract:
The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings. After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2), it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development.read more
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Book ChapterDOI
Construction of Human Antibody Gene Libraries and Selection of Antibodies by Phage Display
Thomas Schirrmann,Michael Hust +1 more
TL;DR: Methods for the construction of human scFv gene libraries and the antibody selection are described, which directly allow the selection of human antibodies and the corresponding genes from human antibody gene libraries.
Journal ArticleDOI
Improved microtitre plate production of single chain Fv fragments in Escherichia coli.
Michael Hust,Miriam Steinwand,Laila Al-Halabi,Saskia Helmsing,Thomas Schirrmann,Stefan Dübel +5 more
TL;DR: A combined protocol was developed which improved the expression of scFv fragments over standard methods, and was not possible to deduce a single set of optimal parameters applicable to all the tested scFvs.
Book ChapterDOI
Selection of Recombinant Antibodies From Antibody Gene Libraries
TL;DR: Detailed protocols are given for the selection of recombinant antibody fragments from antibody gene libraries in microtiter plates based on the specific molecular interaction of antibody phage with an immobilized antigen thus allowing the enrichment and isolation of antigen-specific monoclonal binders from very large antibody Gene libraries.
Journal ArticleDOI
Exploiting sequence and stability information for directing nanobody stability engineering.
Patrick Kunz,Tilman Flock,Nicolas Soler,Moritz Zaiss,Cécile Vincke,Yann G.-J. Sterckx,Damjana Kastelic,Serge Muyldermans,Jörg D. Hoheisel +8 more
TL;DR: The potential and limitations of engineering nanobody thermostability are illustrated by merging sequence information with stability data, an aspect that is becoming increasingly important with the recent development of high-throughput biophysical methods.
References
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