Sustained high-yield production of recombinant proteins in transiently transfected COS-7 cells grown on trimethylamine-coated (hillex) microcarrier beads.
The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.
Knibbs
Randall N
RN
Department of Pathology, University of Michigan, Ann Arbor 48109-0602, USA.
Dame
Michael
M
Allen
Melissa R
MR
Ding
Yunhong
Y
Hillegas
William J
WJ
Varani
James
J
Stoolman
Lloyd M
LM
D000818:Animals; D019556:COS Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002522:Chlorocebus aethiops / Q000235:genetics; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis / Q000494:pharmacology*; D019040:E-Selectin / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D007075:Immunoglobulin M / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008744:Methylamines / Q000494:pharmacology; D051379:Mice / Q000235:genetics; D008863:Microspheres; D019007:P-Selectin / Q000096:biosynthesis / Q000235:genetics; D010316:Particle Size; D011137:Polystyrenes / Q000494:pharmacology; D015202:Protein Engineering / Q000379:methods; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection / Q000379:methods*
D020099:Coated Materials, Biocompatible; D019040:E-Selectin; D007075:Immunoglobulin M; D008164:Luminescent Proteins; D008744:Methylamines; D019007:P-Selectin; D011137:Polystyrenes; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C023336:trimethylamine