Mammalian expression of isotopically labeled proteins for NMR spectroscopy.
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.
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3027, USA. sastrym@mail.nih.gov
Mallika
M
Sastry
Carole A
CA
Bewley
Peter D
PD
Kwong
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry
D011994:Recombinant Proteins