See Structural basis of Wnt recognition by Frizzled, Janda et.al. 2012, for full details.
Recombinant expression
The xWnt8 gene was cloned into a Drosophila vector pACTIN-SV.
The PROMOTER was Drosophila actin 5c.
The SIGNAL SEQUENCE used was an artificial human sequence of CD8 alpha chain.
The PROTEASE CLEAVAGE SITE was a 3C C-terminal LEVLFQ/GP.
TAGS include Fc-tag and 6His.
A filtration column - A-Sepharose Fast flow (Sigma) column.
Preparation of selenomethionine-labelled xWnt-Fz8-CRD complex
Insect express medium S2 cells were grown that expressed xWnt and Fz8-CRD-Fc. The growth mediums were switched to incorporate selenomethionine.
Recombinant Fz-CRD for yeast expression display and chip immobilization
The CRD (cysteine-rich-domain) of human Fz4 and 5, plus mouse Fz8 - with C-terminal protease site, a BAP tag for accepting biotin, and 6His tag were expressed in the baculovirus expresson system. (Plasmid = pAcGP67-A vector).
Purification was carried out using Nickel affinity chromatography and size-based chromatography.
Biotin was also added.
Mini-xWnt8 expression
Residues 248-338 from the C-terminus of xWnt8 (with a 6His tag) were expressed in the pAcGP67-A vector and expressed in the same way as the recombinantFz-CRD.
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