High-resolution heteronuclear multidimensional NMR of proteins in living insect cells using a baculovirus protein expression system

Hamatsu, J. et al. (2013) High-resolution heteronuclear multidimensional NMR of proteins in living insect cells using a baculovirus protein expression system. Journal of the American Chemical Society, 135(5), pp. 1688-1691. (doi:10.1021/ja310928u)

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Abstract

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. In order to complement the existing protocols, and to extend the range of possible applications, we introduce a novel approach for observing in-cell NMR spectra using the sf9 cell/baculovirus system. High-resolution 2D 1H–15N correlation spectra were observed for four model proteins expressed in sf9 cells. Furthermore, 3D triple-resonance NMR spectra of the Streptococcus protein G B1 domain were observed in sf9 cells by using nonlinear sampling to overcome the short lifetime of the samples and the low abundance of the labeled protein. The data were processed with a quantitative maximum entropy algorithm. These were assigned ab initio, yielding approximately 80% of the expected backbone NMR resonances. Well-resolved NOE cross peaks could be identified in the 3D 15N-separated NOESY spectrum, suggesting that structural analysis of this size of protein will be feasible in sf9 cells.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Smith, Dr Brian
Authors: Hamatsu, J., O’Donovan, D., Tanaka, T., Shirai, T., Hourai, Y., Mikawa, T., Ikeya, T., Mishima, M., Boucher, W., Smith, B.O., Laue, E.D., Shirakawa, M., and Ito, Y.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Journal of the American Chemical Society
Publisher:American Chemical Society
ISSN:0002-7863
ISSN (Online):1520-5126
Published Online:18 January 2013

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