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 microg of protein. Moreover, large numbers of constructs can be generated, screened and scaled up for expression and crystallization, with results obtained in 4 weeks.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Lee JE,Fusco ML,Saphire EOdoi
10.1038/nprot.2009.29subject
Has Abstractpub_date
2009-01-01 00:00:00pages
592-604issue
4eissn
1754-2189issn
1750-2799pii
nprot.2009.29journal_volume
4pub_type
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