Construction of protein analogues by site-specific condensation of unprotected fragments.
The extreme sensitivity to periodate of 1-amino, 2-hydroxy compounds permits the selective conversion of N-terminal serine and threonine to an aldehydic group. We have used this reaction to construct analogues of human granulocyte colony stimulating factor (G-CSF) by allowing such oxidized peptides to react with others that have had a hydrazide derivative attached to the C-terminus by reversed proteolysis. Two recombinant analogues of G-CSF were used as starting materials. Both had only a single lysine residue (at position 62 and 75, respectively) followed immediately by a serine. Digestion of each analogue by the lysine-specific protease from Achromobacter lyticus gave two fragments, one of which could be N-terminally oxidized and the other converted to the C-terminal hydrazide derivative by reversed proteolysis using the same enzyme. After preliminary studies with model peptides, we first reacted the corresponding peptide pairs together and then, in order to eliminate the 64-74 disulfide loop, fragment 1-62 from the first analogue with fragment 76-174 from the second. Reactions are efficient (up to 80% product based on the oxidized fragment) and take place under very mild conditions. The hydrazone bond can easily be stabilized by reduction with NaBH3CN. This method represents a new, reasonably general route for the construction of large protein chimeras of precisely controlled structure.
Gaertner
H F
HF
Département de Biochimie Médicale, Centre Médical Universitaire, Geneva, Switzerland.
Rose
K
K
Cotton
R
R
Timms
D
D
Camble
R
R
Offord
R E
RE
D000595:Amino Acid Sequence; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000235:genetics; D006801:Humans; D006835:Hydrazones; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis; D011506:Proteins / Q000138:chemical synthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis
D006835:Hydrazones; D007202:Indicators and Reagents; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor