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An efficient platform for screening expression and crystallization of glycoproteins produced in human cells

Nature Protocols volume 4, pages 592–604 (2009)Cite this article

Abstract

Glycoproteins are involved in diverse biological processes ranging from extracellular contact and recognition to intracellular signaling. Crystal structures of glycoproteins would yield tremendous insight into these processes. But glycoprotein structural analysis has been hindered by difficulties in expressing milligram quantities of stable, homogeneous protein and determining which modifications will yield samples amenable to crystallization. We describe a platform, which we have proven to be effective for rapidly screening expression and crystallization of a challenging glycoprotein target. In this protocol, multiple glycoprotein ectodomain constructs are produced in parallel by transient expression of adherent human embryonic kidney (HEK) 293T cells and are subsequently screened for crystals in microscale quantities by free interface diffusion. As a result, recombinant proteins are produced and processed in a native, mammalian environment, and crystallization screening can be accomplished with as little as 65 μg of protein. Moreover, large numbers of constructs can be generated, screened and scaled up for expression and crystallization, with results obtained in 4 weeks.

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Figure 1: Workflow for the HEK293T-microfluidic crystallization screening platform.
Figure 2: Large-scale expression.
Figure 3: Substrate specificities of selected glycosidases useful for large-scale deglycosylation.
Figure 4: Microfluidic crystallization chip.
Figure 5: Crystallization using microfluidic free interface diffusion.
Figure 6: Test expression and deglycosylation of glycoproteins.

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Acknowledgements

We thank Dr Robyn Stanfield at The Scripps Research Institute for assistance with the Fluidigm microfluidic system and members of the Ollmann Saphire laboratory for advice and help. This study was supported by NIH NIAID operating grants (AI053423 and AI067927) and a Career Award by the Burroughs Wellcome Fund to E.O.S. and a fellowship to J.E.L. from the Canadian Institutes of Health Research. This is manuscript no. 19626 from The Scripps Research Institute.

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Author notes

  1. Jeffrey E Lee and Marnie L Fusco: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    Jeffrey E Lee, Marnie L Fusco & Erica Ollmann Saphire

  2. The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    Erica Ollmann Saphire

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Correspondence to Erica Ollmann Saphire.

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Lee, J., Fusco, M. & Saphire, E. An efficient platform for screening expression and crystallization of glycoproteins produced in human cells. Nat Protoc 4, 592–604 (2009). https://doi.org/10.1038/nprot.2009.29

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