Protein Purification Using PDZ Affinity Chromatography

Abstract

PDZ domains function in nature as protein‐binding domains within scaffold and membrane‐associated proteins. They comprise approximately 90 residues and undergo specific, high‐affinity interactions with complementary C‐terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain‐binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. © 2015 by John Wiley & Sons, Inc.

A model for regulation by SynGAP-α1 of binding of synaptic proteins to PDZ-domain 'Slots' in the postsynaptic density

“…SynGAP-α1 can be expressed in bacteria and purified in a soluble form by deleting the first 102 residues of its N-terminus ( Walkup et al, 2015 ). This version of synGAP, termed r-synGAP-α1, retains all of the identified functional domains, the regulatory domain, and the C-terminal PDZ ligand ( Figure 2A ).…”

“… The GAP activity of r-synGAP-α1 (250 nM) for the indicated GTP-binding protein was assayed as described in Walkup et al (2015) except that 1 mM Ca 2+ , 3.4 µM CaM, or both were added to the GAP assay. A, HRas GAP activity; B, Rap1 GAP activity; and C, Rap2 GAP activity.…”

“…Similarly, the change in protein composition of PSDs in Syngap +/- animals ( Figure 11 ) cannot be explained by a two-fold reduction in RasGAP and RapGAP activity. SynGAP produces a much larger fold increase in GTPase activity of Rap than of Ras ( Walkup et al, 2015 and references therein). Therefore, loss of half of the GAP activity would be predicted to increase the level of active Rap even more than that of active Ras.…”

“…Soluble recombinant PDZ domains, comprising residues 61–151 (PDZ1), 155–249 (PDZ2), 302–402 (PDZ3), 61–249 (PDZ12), and 61–403 (PDZ123) from murine PSD-95 (Q62108) were purified from E. coli as previously described ( Walkup and Kennedy, 2014 , 2015 ) with the modifications below.…”

“…PDZ domain affinity resins (PDZ1, residues 61–151; PDZ2, residues 155–249; PDZ3, residues 302–402; PDZ12, residues 61–249; PDZ123, residues 61–402 from murine PSD-95) were prepared by the HaloTag-HaloLink method as previously described ( Walkup and Kennedy, 2014 , 2015 ). Briefly, bacterial cell pellets containing PDZ domain-HaloTag fusion proteins were resuspended in 10 ml/g of Purification Buffer, and lysed by three passes through a ML-110 microfluidizer.…”