Transfection of partially purified plasmid DNA for high level transient protein expression in HEK293-EBNA cells.
One of the major constraints to performing large-scale transfections of cultured mammalian cells for the transient expression of recombinant proteins is the production of large quantities of purified plasmid DNA. In this report partially purified plasmid DNA was prepared by a method that combines alkaline lysis of E. coli with standard precipitation techniques. The efficiency of calcium phosphate-DNA co-precipitate formation with crude DNA was similar to that observed for pure DNA, but precipitate formed with crude DNA also contained RNA. The transfection of adherent and suspension-adapted HEK293-EBNA cells with partially purified pEGFPN1 resulted in levels of transient GFP expression equivalent to those achieved with pure DNA. In addition, the co-transfection of 1-200 ml cultures of suspension-adapted HEK293-EBNA cells with two different plasmids encoding the heavy and light chain genes of anti-human RhD IgG1, respectively, yielded similar IgG titers with pure and partially purified plasmid DNA. Finally, it was observed that suspension-adapted cells were more tolerant to the presence of RNA in the plasmid preparations than were adherent cells. These findings are relevant to the field of DNA transfection, including applications ranging from high-throughput screening to large-scale transient protein expression.
Laboratory of Cellular Biotechnology, Faculty of Basic Sciences, Swiss Federal Institute of Technology Lausanne, 1015 Lausanne, Switzerland.
J L
JL
Wright
M
M
Jordan
F M
FM
Wurm
D019149:Bioreactors; D002130:Calcium Phosphates / Q000378:metabolism; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D010865:Pilot Projects; D010957:Plasmids / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D012204:Rh-Hr Blood-Group System / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection / Q000379:methods*
D002130:Calcium Phosphates; D019309:Epstein-Barr Virus Nuclear Antigens; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D049452:Green Fluorescent Proteins; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous