Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


HEUERMANN

Stable expression of epitope-tagged proteins in mammalian cells

Kenneth E. Heuermann and Bill L. Brizzard

SIGMA Chemical Company, St. Louis, Missouri, USA

Analysis of gene function often requires stable expression of the recombinant gene in a mammalian cell line.  This can be facilitated by incorporating an epitope tag, such as the FLAG® peptide (AspTyrLysAspAspAspAspLys), commonly used for the isolation, purification, and detection of recombinant proteins expressed in E.coli.  pFLAG-CMV-3 and pFLAG-CMV-4 vectors stably express secreted or intracellular N-terminal FLAG-fusion proteins, respectively, in mammalian cell lines. Initially, COS7 cells transiently transfected with pFLAG-CMV-3-BAP or pFLAG-CMV-4-BAP were shown to express bacterial alkaline phosphatase (E. coli phoA) by immunostaining using the M2 anti-FLAG monoclonal antibody.  Western analyses of cell extracts and media confirmed these results.  COS7 and CHO-K1 cells were then transfected with pFLAG-CMV-3, pFLAG-CMV-3-BAP, pFLAG-CMV-4, or pFLAG-CMV-4-BAP, to obtain stably transformed cell lines.  Transient expression of BAP by cells transfected with pFLAG-CMV-3-BAP or pFLAG-CMV4-BAP, but not pFLAG-CMV-3 or pFLAG-CMV-4, was demonstrated by immunostaining in parallel test plates.  Transfected cells were then selected by treatment with 500 ug/ml G418 sulfate.  At 20 days post-transfection after three changes of medium, surviving COS7 and CHO cells transfected with pFLAG-CMV-3-BAP or pFLAG-CMV-4-BAP continued to express BAP, as shown by detection of FLAG-tagged BAP by Western analysis. This result indicates stable integration of the neomycin resistance gene and FLAG-tagged BAP construct.


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