High-Resolution Heteronuclear Multidimensional NMR of Proteins in Living Insect Cells Using a Baculovirus Protein Expression System

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• 作者：Jumpei Hamatsu ; Daniel O鈥橠onovan ; Takashi Tanaka ; Takahiro Shirai ; Yuichiro Hourai ; Tsutomu Mikawa ; Teppei Ikeya ; Masaki Mishima ; Wayne Boucher ; Brian O. Smith ; Ernest D. Laue ; Masahiro Shirakawa ; Yutaka Ito
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：February 6, 2013
• 年：2013
• 卷：135
• 期：5
• 页码：1688-1691
• 全文大小：329K
• 年卷期：v.135,no.5(February 6, 2013)
• ISSN：1520-5126

文摘

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鈥?sup>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.