Extended Data Fig. 3: Purification of recombinant YG-domain polypeptides used to prepare YG hydrogels.
From: Import mechanism of peroxisomal proteins with an N-terminal signal sequence
a, Left, scheme depicting the domain organization of PEX13 from Arabidopsis thaliana. Note that A. thaliana PEX13 lacks the transmembrane segment and SH3 domain found in metazoan PEX13. Relevant residue numbers are specified on top, and the region that was produced recombinantly to prepare YG hydrogels is underlined beneath. The recombinant fragment was fused at the N terminus to a tetradecahistidine (14×His) tag and a TEV protease-cleavage site, and at the C terminus to a cysteine (Cys) residue. Right, purification of the wild-type fragment (WT) and the version lacking the KPWE motif (∆KPWE). Lanes 1 and 5 show each His-tagged precursor purified by nickel-affinity chromatography in the presence of urea (as described in the Methods), resolved by SDS–PAGE and stained by Coomassie blue. Lanes 2 and 6 show each fragment after cleavage with TEV protease to release the His tag. Lanes 3 and 7 show the soluble fraction (sup) of each TEV-cleavage reaction, demonstrating that each fragment remains soluble in urea in the absence of the tag. The cleaved tag was removed from the reactions by a subsequent nickel-affinity step, and the resulting flow-through containing each final purified fragment is shown in lanes 4 and 8. Molecular weights (in kD) are marked on the side; an image of the unprocessed gel is available in source data. b, Thiol-reactive beads were conjugated to the purified wild-type (WT) YG-domain polypeptide or the version lacking the KPWE motif (∆KPWE) described in panel a. The beads were then incubated with PEX7 fused at the C terminus to GFP (PEX7–GFP) or with GFP alone, and binding of each fluorescent protein to the immobilized fragments was visualized on a spinning-disk confocal microscope. Scale bar equals 100 µm.
