title: Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.
abstract: Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).
authors.affiliation: Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Wilke
authors.forename: Lothar
authors.initials: L
authors.lastname: Groebe
authors.forename: Vitali
authors.initials: V
authors.lastname: Maffenbeier
authors.forename: Volker
authors.initials: V
authors.lastname: Jäger
authors.forename: Manfred
authors.initials: M
authors.lastname: Gossen
authors.forename: Jörn
authors.initials: J
authors.lastname: Josewski
authors.forename: Agathe
authors.initials: A
authors.lastname: Duda
authors.forename: Lilia
authors.initials: L
authors.lastname: Polle
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Owens
authors.forename: Dagmar
authors.initials: D
authors.lastname: Wirth
authors.forename: Dirk W
authors.initials: DW
authors.lastname: Heinz
authors.forename: Joop
authors.initials: J
authors.lastname: van den Heuvel
authors.forename: Konrad
authors.initials: K
authors.lastname: Büssow
mesh_terms: D000818:Animals; D055592:Biophysical Phenomena; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line / Q000166:cytology*; D006224:Cricetinae; D003412:Cricetulus; D003460:Crystallization; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry*; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D004254:DNA Nucleotidyltransferases; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein; C045119:FLP recombinase