An efficient platform for screening expression and crystallization of glycoproteins produced in human cells.
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.
Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Jeffrey E
JE
Lee
Marnie L
ML
Fusco
Erica Ollmann
EO
Saphire
D015153:Blotting, Western; D018929:Cell Culture Techniques; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D006023:Glycoproteins; D011993:Recombinant Fusion Proteins