Chapter title |
Mammalian expression of isotopically labeled proteins for NMR spectroscopy.
|
---|---|
Chapter number | 11 |
Book title |
Isotope labeling in Biomolecular NMR
|
Published in |
Advances in experimental medicine and biology, January 2012
|
DOI | 10.1007/978-94-007-4954-2_11 |
Pubmed ID | |
Book ISBNs |
978-9-40-074953-5, 978-9-40-074954-2
|
Authors |
Sastry, Mallika, Bewley, Carole A, Kwong, Peter D, Mallika Sastry, Carole A. Bewley, Peter D. Kwong, Bewley, Carole A., Kwong, Peter D. |
Abstract |
NMR spectroscopic characterization of biologically interesting proteins generally requires the incorporation of (15)N/(13)C and/or (2)H stable isotopes. While prokaryotic incorporation systems are regularly used, mammalian ones are not: of the nearly 9,000 NMR macromolecular structures currently deposited in the Protein Data Bank, only a handful (<0.5%) were solved with proteins expressed in mammalian systems. This low number of structures is largely a reflection of the difficulty in producing uniformly labeled, mammalian-expressed proteins. This is unfortunate, as many interesting proteins require mammalian cofactors, chaperons, or post-translational modifications such as N-linked glycosylation, and mammalian cells have the necessary machinery to produce them correctly. Here we describe recent advances in mammalian expression, including an efficient adenoviral vector-based system, for the production of isotopically enriched proteins. This system allows for the expression of mammalian proteins and their complexes, including proteins that require post-translational modifications. We describe how this system can produce isotopically labeled (15)N and (13)C post-translationally modified proteins, such as the outer domain of HIV-1 gp120, which has 15 sites of N-linked glycosylation. Selective amino-acid labeling is also described. These developments should reduce barriers to the determination of NMR structures with isotopically labeled proteins from mammalian expression systems. |
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