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Studying protein interactions with gBlocks Gene Fragments

Schneider PN, Slusarski DC, Houston DW. (2012) Differential role of axin RGS domain function in Wnt signaling during anteroposterior patterning and maternal axis formation. PLoS One, 7(9):e44096.

Citation summary: Read how scientists use a fragment of adenomatous polyposis coli protein (APC), expressed from a gBlocks Gene Fragment, to monitor interactions of an axin mutant and wild-type RGS domain with this protein. The results provide a better understand of axin’s role in the β-catenin destruction complex, part of a Wnt signaling pathway.

Axin is part of a multi-protein complex that facilitates the phosphorylation and degradation of β-catenin, a critical transcription regulator involved in development. In the presence of Wnt signaling, axin is removed from its role in the β-catenin destruction complex, and β-catenin is able to enter the nucleus to activate gene transcription. In this paper, the authors investigate an RGS (Regulator of G-protein Signaling) domain within axin to see if it has a larger role in regulating activity of the β-catenin destruction complex. The authors do this by mutating the RGS domain of axin, while leaving the other adenomatous polyposis coli (APC, a protein that helps regulate how cells divide) interactions and degradation functions intact.

The mutant is studied in vitro and in vivo using Xenopus laevis and Danio rerio. To confirm that the axin mutant is still able to interact with APC and the destruction complex, the authors cloned a 459 bp portion of the Xenopus APC producing the axin-interacting region.

The authors were able to easily generate the control Xenopus APC fragment by ordering it as a single gBlocks® Gene Fragment and cloning it into a plasmid containing an HA tag sequence. The expressed fragment was then used to confirm that the wild type and mutant axin RGS domains were able to interact normally with APC.

Published Jan 18, 2013