Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells.
Glycosylation is one of the most common posttranslational modifications of proteins. It has important roles for protein structure, stability and functions. In vivo the glycostructures influence pharmacokinetics and immunogenecity. It is well known that significant differences in glycosylation and glycostructures exist between recombinant proteins expressed in mammalian, yeast and insect cells. However, differences in protein glycosylation between different mammalian cell lines are much less well known. In order to examine differences in glycosylation in mammalian cells we have expressed 12 proteins in the two commonly used cell lines HEK and CHO. The cells were transiently transfected, and the expressed proteins were purified. To identify differences in glycosylation the proteins were analyzed on SDS-PAGE, isoelectric focusing (IEF), mass spectrometry and released glycans on capillary gel electrophoresis (CGE-LIF). For all proteins significant differences in the glycosylation were detected. The proteins migrated differently on SDS-PAGE, had different isoform patterns on IEF, showed different mass peak distributions on mass spectrometry and showed differences in the glycostructures detected in CGE. In order to verify that differences detected were attributed to glycosylation the proteins were treated with deglycosylating enzymes. Although, culture conditions induced minor changes in the glycosylation the major differences were between the two cell lines.
Protein and Cell Sciences, Geneva Research Center, Merck Serono S.A., 9 chemin des Mines, 1202 Geneva, Switzerland.
Amelie
A
Croset
Laurence
L
Delafosse
Jean-Philippe
JP
Gaudry
Christian
C
Arod
Loic
L
Glez
Christophe
C
Losberger
Damien
D
Begue
Ana
A
Krstanovic
Flavie
F
Robert
Francis
F
Vilbois
Laurent
L
Chevalet
Bruno
B
Antonsson
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D014162:Transfection
D011134:Polysaccharides; D011994:Recombinant Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase