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J. Biol. Chem., Vol. 279, Issue 46, 47520-47527, November 12, 2004

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Murine Wnt-1 with an Internal c-myc Tag Recombinantly Produced in Escherichia coli Can Induce Intracellular Signaling of the Canonical Wnt Pathway in Eukaryotic Cells^*

Beatrix Fahnert¶, Johanna Veijola||, Giulietta Roël**, Minna K. Kärkkäinen, Antti Railo, Olivier Destrée**, Seppo Vainio, and Peter Neubauer

From the Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, P. O. Box 4300, FIN-90014 Oulu, Finland, the Biocenter Oulu, P. O. Box 5000, FIN-90014 Oulu, Finland, the ^**Netherlands Institute for Developmental Biology, Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands, and the Department of Biochemistry, University of Oulu, P. O. Box 3000, FIN-90014 Oulu, Finland

Wnt-1 belongs to the Wnt family of secreted glycoproteins inducing an intracellular signaling pathway involved in cell proliferation, differentiation, and pattern formation. The canonical branch is one of three known branches. This is also valid in vitro, and Wnts can be considered beneficial for culturing primary cells from organs, provided Wnts are available and applicable even with cells of different species. It was shown here that internally c-myc-tagged murine Wnt-1 produced in the heterologous host Escherichia coli was appropriate for inducing intracellular signaling of the canonical Wnt pathway in eukaryotic cells via stabilization of cytosolic -catenin. The pioneering injection of the protein into the blastocoels of Xenopus laevis embryos led to axis duplication and suppression of head formation. Applying the recombinant murine Wnt-1 to metanephric mesenchyme activated the tubulogenic program. The signalinducing activity of the recombinant protein was also positively demonstrated in the TOPflash reporter assay. Although Wnts were purified recently from the growth media of stably transfected eukaryotic cell lines, the production of active Wnt proteins in pro- or eukaryotic microorganisms reportedly has never been successful. Here soluble production in E. coli and translocation into the oxidizing environment of the periplasm were achieved. The protein was purified using the internal c-myc tag. The effect on the eukaryotic cells implies that activity was retained. Thus, this approach could make recombinant murine Wnt-1 available as a good starting point for other Wnts needed, for example, for maintaining and differentiating stem cells, organ restoration therapy, and tissue engineering.

Received for publication, March 23, 2004 , and in revised form, August 25, 2004.

^* This work was supported by a Marie Curie Fellowship of the European Community program "Quality of Life Individual Fellowships of the Fifth Framework Programme" under Contract QLK3-CT-2001-51066 (to B. F.), by Academy of Finland Project 52477, and by European Union Research, Technology, and Development Project QLK3-CT2001-01275. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Present address: Clinical Research Center, University of Oulu, P. O. Box 5000, FIN-90014 Oulu, Finland.

^¶ To whom correspondence should be addressed. Tel.: 358-8-5532361; Fax: 358-8-5532304; E-mail: beatrix.fahnert{at}oulu.fi.

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