Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development

Tools

Item Type:	Article
Title:	Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development
Creators Name:	Wilke, S., Groebe, L., Maffenbeier, V., Jaeger, V., Gossen, M., Josewski, J., Duda, A., Polle, L., Owens, R.J., Wirth, D., Heinz, D.W., van den Heuvel, J. and Buessow, K.
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).
Keywords:	Biophysical Phenomena, CHO Cells, Cell Culture Techniques, Cell Line, Cricetinae, Cricetulus, Crystallization, DNA Nucleotidyltransferases, Genetic Recombination, Genetic Vectors, Glycoproteins, Glycosylation, Green Fluorescent Proteins, Luminescent Proteins, Recombinant Proteins, Tertiary Protein Structure, Animals
Source:	PLoS ONE
ISSN:	1932-6203
Publisher:	Public Library of Science
Volume:	6
Number:	12
Page Range:	e27829
Date:	December 2011
Official Publication:	https://doi.org/10.1371/journal.pone.0027829
PubMed:	View item in PubMed

Repository Staff Only: item control page