Cell-surface streptavidin fusion protein for rapid selection of transfected mammalian cells.
We developed a series of eight mammalian cell surface marker fusion genes by using the streptavidin gene from Streptomyces avidinii. These fusion genes are useful and non-growth-toxic selection markers for rapid-harvest transfected mammalian cells. Two streptavidin constructs were used; the longer fragment contains the native bacterial signal sequence, which the shorter fragment lacks. For expression of the streptavidin antigen on the surface of mammalian cells, streptavidin was flanked by a mammalian signal sequence and a transmembrane domain (from mouse H2-K or Kit); some constructs also contained the gene for enhanced green fluorescent protein (EGFP). We transfected a series of plasmids encoding the fusion proteins into HeLa cells and determined that the transfected cells produced the fusion protein on their cell surfaces. To separate transfected cells from nontransfected cells, we incubated cells with a polyclonal antibody against streptavidin, and antibody-bound cells were harvested by the use of paramagnetic beads coupled with the corresponding secondary antibody. We obtained highly pure populations of transfected cells; this result also confirmed the production of the fusion protein on the cell surface. Cell growth assays revealed that none of the transfected fusion genes or their products adversely affected the proliferation of HeLa cells. Our results indicate that the fusion constructs we developed and the immunomagnetic separation protocol we used are valuable tools for various transfection applications. In particular, the constructs containing EGFP are advantageous because transfection efficiency can be assessed without additional treatment of cells.
Gotoh
Hideo
H
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan. gotoh@affrc.go.jp
Matsumoto
Yukiko
Y
D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular; D003584:Cytological Techniques / Q000379:methods*; D005819:Genetic Markers; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D018189:Immunomagnetic Separation; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D019809:Streptavidin / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D005819:Genetic Markers; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019809:Streptavidin