Effects of pCIneo and pCEP4 expression vectors on transient and stable protein production in human and simian cell lines |
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Authors: | Janet H Parham Tom Kost Jeff T Hutchins |
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Institution: | (1) GlaxoSmithKline Discovery Research, 5 Moore Drive, Research Triangle Park, NC 27709, USA;(2) GlaxoSmithKline Discovery Research, 5 Moore Drive, Research Triangle Park, NC 27709, USA |
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Abstract: | To support and meet the demand for recombinant proteins early in the drug discovery process, much work has been directed toward
improving the methods used for transient gene transfection and expression. A factor which could potentially affect the outcome
of experiments is the choice of the expression vector. Conventional vectors such as pCIneo and pcDNA3 have been used frequently.
Each of these places the gene of interest under the control of the CMV promoter. An interesting alternative is provided by
episomal vectors. For example, the pCEP4 vector contains the gene coding for the Epstein Barr nuclear antigen as well as the
EBNA ori P sequence. This combination allows for the episomal replication of the plasmid. In preliminary experiments, we compared
transient secreted placental alkaline phosphatase production in 8 cell lines from 3 different species using the pCIneo vs.
pCEP4 vectors and found the utility of the pCEP4 vector to be limited to the human 293 EBNA cell line. In this paper, we have
compared the two vectors in six cell lines of simian and human origin, measuring the transient production of secreted placental
alkaline phosphatase and human hepatocyte growth factor. In general, the pCEP4 vector produced higher amounts of both proteins
in transient transfections. Results were particularly pronounced in the HEK 293 and 293 EBNA cell lines. Stable pools of cells
(uncloned) expressing human hepatocyte growth factor were isolated using pCIneo and pCEP4 and protein production levels were
compared to those seen in transient transfections. Stable expression with pCEP4 was found to produce the highest levels of
human hepatocyte growth factor in 3 of 4 cell lines. Finally, electroporation and FuGENETM6(Roche, Indianapolis IN) as transfection methods were compared measuring transient production of secreted placental alkaline
phosphatase, human hepatocyte growth factor, and green fluorescent protein. FuGENE produced higher protein concentrations
in less time than electroporation for all 3 proteins.
This revised version was published online in July 2006 with corrections to the Cover Date. |
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Keywords: | episomal vectors Epstein Barr nuclear antigen human hepatocyte growth factor secreted placental alkaline phosphatase transfection |
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