Glycoprotein production for structure analysis with stable, glycosylation mutant CHO cell lines established by fluorescence-activated cell sorting.
Stable mammalian cell lines are excellent tools for the expression of secreted and membrane glycoproteins. However, structural analysis of these molecules is generally hampered by the complexity of N-linked carbohydrate side chains. Cell lines with mutations are available that result in shorter and more homogenous carbohydrate chains. Here, we use preparative fluorescence-activated cell sorting (FACS) and site-specific gene excision to establish high-yield glycoprotein expression for structural studies with stable clones derived from the well-established Lec3.2.8.1 glycosylation mutant of the Chinese hamster ovary (CHO) cell line. We exemplify the strategy by describing novel clones expressing single-chain hepatocyte growth factor/scatter factor (HGF/SF, a secreted glycoprotein) and a domain of lysosome-associated membrane protein 3 (LAMP3d). In both cases, stable GFP-expressing cell lines were established by transfection with a genetic construct including a GFP marker and two rounds of cell sorting after 1 and 2 weeks. The GFP marker was subsequently removed by heterologous expression of Flp recombinase. Production of HGF/SF and LAMP3d was stable over several months. 1.2 mg HGF/SF and 0.9 mg LAMP3d were purified per litre of culture, respectively. Homogenous glycoprotein preparations were amenable to enzymatic deglycosylation under native conditions. Purified and deglycosylated LAMP3d protein was readily crystallized. The combination of FACS and gene excision described here constitutes a robust and fast procedure for maximizing the yield of glycoproteins for structural analysis from glycosylation mutant cell lines.
Division of Structural Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
Sonja
S
Wilke
Joern
J
Krausze
Manfred
M
Gossen
Lothar
L
Groebe
Volker
V
Jäger
Ermanno
E
Gherardi
Joop
J
van den Heuvel
Konrad
K
Büssow
D000818:Animals; D016466:CHO Cells; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods*; D058851:GPI-Linked Proteins; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D015816:Cell Adhesion Molecules, Neuronal; D058851:GPI-Linked Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C095362:limbic system-associated membrane protein; C082332:macrophage stimulating protein; D049452:Green Fluorescent Proteins; D017228:Hepatocyte Growth Factor