Received for publication April 8, 2005.
Revised June 2, 2005.
Accepted for publication June 2, 2005.

The reduced folate carrier gene is a novel selectable marker for recombinant protein overexpression

Abstract

Folate cofactors are one-carbon donors essential for the biosynthesis of purines and thymidylate. Mammalian cells are devoid of folate biosynthesis and are therefore folate auxotrophs which take up folate vitamins primarily via the reduced folate carrier (RFC). Here we show that the human RFC (hRFC) gene can serve as a novel selectable marker for the overproduction of recombinant proteins. Toward this end, a hemagglutinin (HA) epitope tagged hRFC (hRFC-HA) was introduced into a bicistronic vector (pIRES2-EGFP), upstream of an enhanced green fluorescent protein (EGFP) reporter gene. Chinese hamster ovary cells deficient in RFC activity were isolated and transfected with this construct, followed by gradual deprivation of leucovorin, the sole folate source in the growth medium. Only cells with hRFC-HA overexpression were able to take up leucovorin and thereby survive these selective conditions. Western blot and immunofluorescence analyses confirmed that the hRFC-HA was overexpressed at extremely high levels, properly glycosylated and sorted out to the plasma membrane. This resulted in a ~ 450-fold increased [³H]methotrexate influx and ~100-fold increased sensitivity to methotrexate, relative to untransfected RFC-deficient cells. Consistently, flow cytometric analysis revealed that EGFP was overexpressed ~100-fold above the autofluorescence level. Overproduction of hRFC-HA and EGFP was stably maintained for at least two months in a constant concentration of leucovorin. These results establish a novel RFC-based metabolic selection system for the efficient overexpression of recombinant proteins. Furthermore, possible implications to sub-cellular transporter localization and restoration of MTX sensitivity in drug resistant tumors by RFC-based gene therapy are being discussed.

Key words: Ion transporters (SERCA, Na/K ATPase, CFTR), Overexpression

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