Purification of the PACAP-1 receptor by ligand affinity chromatography

Employing the Fmoc solid phase strategy, analogues of pituitary adenylate cyclase activating polypeptide (PACAP) were synthesized containing single cysteine residues. Monobiotinylation of these analogues was achieved via thioether formation between the sulfhydryl groups provided by the cysteine residues and the biotinylation reagent N-lodoacetyl-N'biotinyl-hexylenediamine. Almost all of the S-biotinylated analogues revealed full binding activity to the solubilized PACAP-1 receptor from pig brain membranes. To minimize sterical hindrance of ternary complex formation between the biotinylated analogues, the PACAP-1 receptor and streptavidin, an analogue was designed which contains seven consecutive alanine residues between position 28 and 34. This biotinylated analogue, S-biotinyl[Ala28-34,Cys35]PACAP(1-35), was highly capable of ternary complex formation and therefore used as high affinity ligand for affinity chromatography. By means of lectin adsorption chromatography and ligand affinity chromatography the PACAP-1 receptor was purified more than 6000-fold from porcine brain membranes solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonate (CHAPSO).