Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells : peptone additives improve cell growth and transfection efficiency

Download
  1. Get@NRC: Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells : peptone additives improve cell growth and transfection efficiency (Opens in a new window)
DOIResolve DOI: http://doi.org/10.1002/bit.10774
AuthorSearch for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for:
TypeArticle
Journal titleBiotechnology and Bioengineering
Volume84
Issue3
Pages332342; # of pages: 11
AbstractLarge-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L-1. To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada; NRC Biotechnology Research Institute
Peer reviewedNo
NPARC number12338306
Export citationExport as RIS
Report a correctionReport a correction
Record identifierb7d73512-5f71-4cbc-8157-1d6b5ee24a26
Record created2009-09-10
Record modified2016-05-09
Bookmark and share
  • Share this page with Facebook (Opens in a new window)
  • Share this page with Twitter (Opens in a new window)
  • Share this page with Google+ (Opens in a new window)
  • Share this page with Delicious (Opens in a new window)