An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts
JournalArticle (Originalarbeit in einer wissenschaftlichen Zeitschrift)
 
ID 3326617
Author(s) Opitz, Christian; Isogai, Shin; Grzesiek, Stephan
Author(s) at UniBasel Grzesiek, Stephan
Isogai, Shin
Opitz, Christian
Year 2015
Title An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts
Journal Journal of Biomolecular NMR
Volume 62
Number 3
Pages / Article-Number 373-385
Abstract Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein (15)N and (13)C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor.
Publisher Springer
ISSN/ISBN 0925-2738 ; 1573-5001
edoc-URL http://edoc.unibas.ch/39972/
Full Text on edoc No
Digital Object Identifier DOI 10.1007/s10858-015-9954-3
PubMed ID http://www.ncbi.nlm.nih.gov/pubmed/26070442
ISI-Number WOS:000357489200012
Document type (ISI) Article
 
   

MCSS v5.8 PRO. 0.709 sec, queries - 0.000 sec ©Universität Basel  |  Impressum   |    
08/08/2020