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.
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 beta-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 signal-inducing activity of the recombinant protein was also positively demonstrated in the TOP-flash 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.
Fahnert
Beatrix
B
Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, P. O. Box 4300, FIN-90014 Oulu, Finland. beatrix.fahnert@oulu.fi
Veijola
Johanna
J
Roël
Giulietta
G
Kärkkäinen
Minna K
MK
Railo
Antti
A
Destrée
Olivier
O
Vainio
Seppo
S
Neubauer
Peter
P
D000818:Animals; D002460:Cell Line; D004622:Embryo, Mammalian / Q000033:anatomy & histology / Q000502:physiology; D004625:Embryo, Nonmammalian; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000502:physiology*; D017930:Genes, Reporter; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009024:Morphogenesis / Q000502:physiology; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D051153:Wnt Proteins; D051155:Wnt1 Protein; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000502:physiology
D036341:Intercellular Signaling Peptides and Proteins; C489428:Myc protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C495249:WNT1 protein, Xenopus; C495247:WNT1 protein, human; D051153:Wnt Proteins; D051155:Wnt1 Protein; C495246:Wnt1 protein, mouse; D029867:Xenopus Proteins