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Mammalian Expression of Isotopically Labeled Proteins for NMR Spectroscopy

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Isotope labeling in Biomolecular NMR

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 992))

Abstract

NMR spectroscopic characterization of biologically interesting proteins generally requires the incorporation of 15N/13C and/or 2H 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 15N and 13C 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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Abbreviations

CHO:

Chinese Hamster Ovary cells

HEK:

Human Embryonic Kidney cells

BHK21:

Baby Hamster Kidney cells

HIV-1:

Human Immunodeficiency Virus Type 1

RSV:

Rous Sarcoma Virus

CMV:

Cytomegalovirus

PEI:

Polyethyleneimine

ITR:

Inverted terminal repeat

BGHpA:

Bovine growth hormone polyadenylation signal

SPR:

Surface plasmon resonance

PBS:

Phosphate buffered saline

ATCC:

American Type Culture Collection

DMEM:

Dubelco’s modified eagle media

FBS:

Dialyzed fetal bovine serum

CAR:

Coxsackie-and Adenovirus Receptor.

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Acknowledgments

We thank the NMR staff at the New York Structural Biology Consortium for assistance with instrumentation and data acquisition. We also thank the members of the Structural Biology Section and Structural Bioinformatics Section at the Vaccine Research Center for insightful comments and discussions. Support for this work was provided by the Intramural Program of the NIH (NIAID and NIDDK). 900 MHz spectrometers were purchased with funds from NIH, USA, the Keck Foundation, New York State, and the NYC Economic Development Corporation.

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Authors and Affiliations

  1. Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 40 Convent Drive; Building 40, Room 2613B, Bethesda, MD, 20892-3027, USA

    Mallika Sastry & Peter D. Kwong

  2. Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA

    Carole A. Bewley

Authors
  1. Mallika Sastry
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  2. Carole A. Bewley
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  3. Peter D. Kwong
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Correspondence to Mallika Sastry .

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Editors and Affiliations

  1. , NMR Research Centre, Indian Institute of Science, Bangalore, India

    Hanudatta S. Atreya

Appendix

Appendix

Table 2 A list of reagents used in the adenoviral based mammalian expression system
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Sastry, M., Bewley, C.A., Kwong, P.D. (2012). Mammalian Expression of Isotopically Labeled Proteins for NMR Spectroscopy. In: Atreya, H. (eds) Isotope labeling in Biomolecular NMR. Advances in Experimental Medicine and Biology, vol 992. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-4954-2_11

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