Efficient mammalian cell expression and single-step purification of extracellular glycoproteins for crystallization

Daniel L. Kober, Zeynep Yurtsever, Thomas J. Brett

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

Original languageEnglish (US)
Article numbere53445
JournalJournal of Visualized Experiments
Issue number106
StatePublished - Dec 23 2015
Externally publishedYes


  • Biochemistry
  • Glycosylation
  • Issue 106
  • Macromolecular crystallography
  • Mammalian protein expression
  • Nickel affinity chromatography

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)


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