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Mammalian Expression of Isotopically Labeled Proteins for NMR Spectroscopy

  • Mallika SastryEmail author
  • Carole A. Bewley
  • Peter D. Kwong
Chapter
Part of the Advances in Experimental Medicine and Biology book series (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.

Keywords

A549 Cell Recombinant Adenovirus Rous Sarcoma Virus Outer Domain Enrich Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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.

Notes

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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Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Mallika Sastry
    • 1
    Email author
  • Carole A. Bewley
    • 2
  • Peter D. Kwong
    • 1
  1. 1.Vaccine Research CenterNational Institute of Allergy and Infectious Diseases, National Institutes of HealthBethesdaUSA
  2. 2.Laboratory of Bioorganic ChemistryNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of HealthBethesdaUSA

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