title: Oxidative folding of synthetic polypeptides S-protected as tert-butylthio derivatives.
abstract: A new method for oxidative folding of synthetic polypeptides assembled by stepwise solid phase synthesis is introduced. Folding is obtained in excellent yields by reacting S-tert-butylthiolated polypeptides with a 100-fold molar excess of cysteine at 37 degrees C in a slightly alkaline buffer containing chaotropic salts, and in the presence of air-oxygen. This novel protocol has been applied to the folding of S-tert-butylthiolated human thymus and activation-regulated chemokine (hu-TARC) derivatives as well as to larger segments of Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins. Folded P. falciparum polypeptides have been used as substrates of endoproteinase Glu-C (Glu-C) and endoproteinase Asp-N (Asp-N) in an attempt to identify their disulfide connectivities. Particular practical advantages of the present method are (i) easy purification and storage of the S-protected peptide derivatives, (ii) elimination of the risk of cysteine alkylation during the acidolytic cleavage deprotection and resin cleavage steps, (iii) possibility to precisely evaluate the extent of folding and disulfide bond formation by mass spectrometry, and (iv) facile recovery of the final folded product.
authors.affiliation: Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland. antonioverdini@yahoo.it
authors.forename: Antonio
authors.initials: A
authors.lastname: Verdini
authors.forename: Silvia
authors.initials: S
authors.lastname: Terenzi
authors.forename: Vincent
authors.initials: V
authors.lastname: Brossard
authors.forename: Mario
authors.initials: M
authors.lastname: Roggero
authors.forename: Giampietro
authors.initials: G
authors.lastname: Corradin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D054414:Chemokine CCL17 / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D010962:Plasmodium berghei / Q000378:metabolism; D010963:Plasmodium falciparum / Q000378:metabolism; D017510:Protein Folding; D015800:Protozoan Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D054414:Chemokine CCL17; D010455:Peptides; D015800:Protozoan Proteins