Preparation of pro-oncogenic mutant forms V659E and V659Q of the transmembrane domain of receptor protein kinase ErbB2 for structural studies

Receptor tyrosine kinases (RTKs) play an important role in intercellular signal transduction through the plasma membrane. RTKs are integral membrane proteins activated upon lateral homo- or heterodimerization involving their transmembrane domain. The polymorphism and mutations in RTK transmembrane (...

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Veröffentlicht in:Biochemistry (Moscow). Supplement series A, Membrane and cell biology Membrane and cell biology, 2013-04, Vol.7 (2), p.91-99
Hauptverfasser: Bocharova, O. V., Bocharov, E. V., Mineev, K. S., Dubinnyi, M. A., Mishin, A. V., Arseniev, A. S.
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container_end_page 99
container_issue 2
container_start_page 91
container_title Biochemistry (Moscow). Supplement series A, Membrane and cell biology
container_volume 7
creator Bocharova, O. V.
Bocharov, E. V.
Mineev, K. S.
Dubinnyi, M. A.
Mishin, A. V.
Arseniev, A. S.
description Receptor tyrosine kinases (RTKs) play an important role in intercellular signal transduction through the plasma membrane. RTKs are integral membrane proteins activated upon lateral homo- or heterodimerization involving their transmembrane domain. The polymorphism and mutations in RTK transmembrane (TM) domains are directly associated with a number of human diseases. The family of epidermal growth factor receptors, ErbB, is an important class of RTKs participating in human cell growth, development, and differentiation. In order to investigate the influence of pathogenic mutations in ErbB TM domains on the structural and dynamic properties of these receptors and on specific interactions of their TM domains, we have developed highly effective systems of bacterial expression and purification of recombinant transmembrane fragments ErbB2 641–684 with pro-oncogenic substitution of Val 659 by Glu or Gln. Transmembrane fragments were obtained in Escherichia coli BL21 (DE3) pLysS as a fusion protein with thioredoxin A. The purification protocol includes immobilized metal ion affinity chromatography (IMAC) and cation-exchange chromatography. The application of the protease Thrombin for hybrid protein hydrolysis considerably reduces financial expenditure as compared to the analogous protocols. The described techniques allow obtaining the milligram quantities of ErbB2 transmembrane fragments and its 15 N-/[ 15 N, 13 C]-isotope-labeled derivatives for the analysis of their spatial structure using high-resolution heteronuclear NMR spectroscopy in a membrane-mimicking milieu.
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1990-7494
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subjects Biomedical and Life Sciences
Cell Biology
Kinases
Life Sciences
Mutation
Proteins
Signal transduction
Spectrum analysis
title Preparation of pro-oncogenic mutant forms V659E and V659Q of the transmembrane domain of receptor protein kinase ErbB2 for structural studies
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