Site-specific religation of G-CSF fragments through a thioether bond.
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.
Gaertner
H F
HF
Département de Biochimie Médicale, Centre Médical Universitaire, Geneva, Switzerland.
Offord
R E
RE
Cotton
R
R
Timms
D
D
Camble
R
R
Rose
K
K
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*
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013440:Sulfides; D016179:Granulocyte Colony-Stimulating Factor