Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development.
PLoS One
; 6(12): e27829, 2011.
Article
en En
| MEDLINE
| ID: mdl-22174749
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).
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Glicoproteínas
/
Línea Celular
/
Técnicas de Cultivo de Célula
/
Fenómenos Biofísicos
Tipo de estudio:
Prognostic_studies
Límite:
Animals
/
Humans
Idioma:
En
Revista:
PLoS One
Asunto de la revista:
CIENCIA
/
MEDICINA
Año:
2011
Tipo del documento:
Article
País de afiliación:
Alemania
Pais de publicación:
Estados Unidos