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Identification of the KRIT1 Protein by LexA-Based Yeast Two-Hybrid System

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Cerebral Cavernous Malformations (CCM)

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2152))

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Abstract

Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system that is associated with leaky capillaries, and a predisposition to serious clinical conditions including intracerebral hemorrhage and seizures. Germline or sporadic mutations in the CCM1/KRIT1 gene are responsible for the majority of cases of CCM. In this article, we describe the original characterization of the CCM1/KRIT1 gene. This cloning was done through the use of a variant of the yeast two-hybrid screen known as the interaction trap, using the RAS-family GTPase KREV1/RAP1A as a bait. The partial clone of KRIT1 (Krev1 Interaction Trapped) initially identified was extended through 5′RACE and computational analysis to obtain a full-length cDNA, then used in a sequential screen to define the integrin-associated ICAP1 protein as a KRIT1 partner protein. We discuss how these interactions are relevant to the current understanding of KRIT1/CCM1 biology, and provide a protocol for library screening with the Interaction Trap.

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Authors and Affiliations

  1. Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, USA

    Ilya G. Serebriiskii, Mohamed Elmekawy & Erica A. Golemis

  2. Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russia

    Ilya G. Serebriiskii

  3. Moscow Institute of Physics and Technology, Moscow Region, Russia

    Mohamed Elmekawy

Authors
  1. Ilya G. Serebriiskii
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  2. Mohamed Elmekawy
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  3. Erica A. Golemis
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Corresponding authors

Correspondence to Ilya G. Serebriiskii or Erica A. Golemis .

Editor information

Editors and Affiliations

  1. Department of Biotechnology, Chemistry and Pharmacy, University of Siena, SIENA, Italy

    Lorenza Trabalzini

  2. Department of Biotechnology, Chemistry and Pharmacy, University of Siena, SIENA, Italy

    Federica Finetti

  3. Department of Clinical and Biological Sciences, School of Medicine and Surgery, University of Torino, ORBASSANO (TORINO), Italy

    Saverio Francesco Retta

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Serebriiskii, I.G., Elmekawy, M., Golemis, E.A. (2020). Identification of the KRIT1 Protein by LexA-Based Yeast Two-Hybrid System. In: Trabalzini, L., Finetti, F., Retta, S. (eds) Cerebral Cavernous Malformations (CCM) . Methods in Molecular Biology, vol 2152. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0640-7_20

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  • DOI: https://doi.org/10.1007/978-1-0716-0640-7_20

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0639-1

  • Online ISBN: 978-1-0716-0640-7

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