title: Site-specific religation of G-CSF fragments through a thioether bond.
abstract: A new approach is described for linking, through a thioether bond, the C-terminus of one unprotected polypeptide with the N-terminus of another. Homocysteine thiolactone is attached to the C-terminus of one polypeptide by reverse proteolysis and provides through hydroxylamine treatment a free sulfhydryl group. The alpha-amino group of a second polypeptide is selectively iodoacetylated by reaction with iodoacetic anhydride at pH 6.0 or the N-hydroxysuccinimide ester derivative at pH 7.0. Coupling of the two modified fragments occurs in a spontaneous alkylation reaction under mild conditions. After preliminary experiments with small peptides, this approach was extended to large protein fragments derived from recombinant analogs of G-CSF by enzymatic digestion. This approach provides a means of making head-to-tail protein chimeras or introducing noncoded structural elements into a protein.
authors.affiliation: Département de Biochimie Médicale, Centre Médical Universitaire, Geneva, Switzerland.
authors.forename: H F
authors.initials: HF
authors.lastname: Gaertner
authors.forename: R E
authors.initials: RE
authors.lastname: Offord
authors.forename: R
authors.initials: R
authors.lastname: Cotton
authors.forename: D
authors.initials: D
authors.lastname: Timms
authors.forename: R
authors.initials: R
authors.lastname: Camble
authors.forename: K
authors.initials: K
authors.lastname: Rose
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013440:Sulfides / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013440:Sulfides; D016179:Granulocyte Colony-Stimulating Factor