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Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family receptors

  • Structural and Functional Analysis of Biopolymers and Their Complexes
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Abstract

ErbB is a family of epidermal growth factor receptors representing an important class of receptor tyrosine kinases that play a leading role in cellular growth, development, and differentiation. Transmembrane domains of these receptors transduce biochemical signals across the plasma membrane via lateral homo- and heterodimerization. The relatively small size of ErbB transmembrane domain complexes with detergents or lipids makes it possible to study their detailed spatial structure using three-dimensional heteronuclear high-resolution NMR spectroscopy. Here, we describe an efficient expression system and a purification procedure for preparative-scale production of transmembrane peptides from all four ErbB proteins—ErbB1, ErbB2, ErbB3, and ErbB4—for the purpose of structural studies. The recombinant peptides were produced in Escherichia coli BL21(DE3)pLysS cells as N-terminal extensions of thioredoxin A. The fusion proteins were cleaved with the light chain of human enterokinase. Several (10–30) milligrams of purified isotope-labeled transmembrane peptides were isolated using a simple and convenient procedure, which consists of consecutive steps of immobilized metal affinity chromatography and cation-exchange chromatography. The purified peptides were reconstituted in a lipid/detergent environment (micelles or bicelles) and characterized using dynamic light scattering and CD and NMR spectroscopy. The data obtained indicate that purified ErbB transmembrane peptides are suitable for structural and dynamic studies of their homo- and heterodimer complexes using high resolution NMR spectroscopy.

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Abbreviations

ErbB:

epidermal growth factor receptor family

DHPC:

dehexanoyl-phosphatidylcholine

DMPC:

dimyristoyl-phosphatidylcholine

DPC:

dodecyl-phosphocholine

IPTG:

isopropyl-β-D-thiogalactoside

RTK:

receptor tyrosine kinase

TM:

transmembrane (protein domain)

IMAC:

immobilized metal affinity chromatography

TrxA:

thioredoxin A

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Authors and Affiliations

  1. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia

    M. V. Goncharuk, A. A. Schulga, Ya. S. Ermolyuk, E. N. Tkach, S. A. Goncharuk, Yu. E. Pustovalova, K. S. Mineev, E. V. Bocharov, I. V. Maslennikov, A. S. Arseniev & M. P. Kirpichnikov

  2. Biological Department, Moscow State University, Moscow, 119991, Russia

    M. V. Goncharuk, S. A. Goncharuk & M. P. Kirpichnikov

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  1. M. V. Goncharuk
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  2. A. A. Schulga
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  3. Ya. S. Ermolyuk
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  4. E. N. Tkach
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  5. S. A. Goncharuk
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  6. Yu. E. Pustovalova
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  7. K. S. Mineev
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  8. E. V. Bocharov
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  9. I. V. Maslennikov
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  10. A. S. Arseniev
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  11. M. P. Kirpichnikov
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Correspondence to M. V. Goncharuk.

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Original Russian Text © M.V. Goncharuk, A.A. Schulga, Ya.S. Ermolyuk, E.N. Tkach, S.A. Goncharuk, Yu.E. Pustovalova, K.S. Mineev, E.V. Bocharov, I.V. Maslennikov, A.S. Arseniev, M.P. Kirpichnikov, 2011, published in Molekulyarnaya Biologiya, 2011, Vol. 45, No. 5, pp. 892–902.

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Goncharuk, M.V., Schulga, A.A., Ermolyuk, Y.S. et al. Bacterial synthesis, purification, and solubilization of transmembrane segments of ErbB family receptors. Mol Biol 45, 823–832 (2011). https://doi.org/10.1134/S0026893311040066

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