Solution synthesis of human midkine, a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five disulphide bonds.
Human midkine (hMK), a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five intramolecular disulphide bonds, was synthesized by solution procedure in order to demonstrate the usefulness of our newly developed solvent system, a mixture of dichloromethane or chloroform and trifluoroethanol. The final protected 121-residue peptide was assembled from two large fully protected intermediates, Boc-(1-59)-OH and H-(60-121)-OBzl, in CHL/TFE(3:1, v/v) using water-soluble carbodiimide in the presence of HOOBt as coupling reagents. After removal of the protecting groups by HF followed by treatment with Hg(OAc)2 in 50% acetic acid, the fully deprotected peptide was subjected to the oxidative folding reaction. The final product was confirmed to have the correct disulphide structure from its tryptic peptide mapping and to possess the same biological activities as those of the natural product. In order to clarify the active region of the hMK molecule, the N-terminal half domains [(1-59) and (60-121)] were also synthesized by the same procedure used for the hMK synthesis. The C-half domain was confirmed to show the full pattern of bioactivities except for the neuronal cell survival activity, while the N-half one showed much less activity in general.
Inui
T
T
Peptide Institute Inc., Protein Research Foundation, Osaka, Japan.
Bódi
J
J
Kubo
S
S
Nishio
H
H
Kimura
T
T
Kojima
S
S
Maruta
H
H
Muramatsu
T
T
Sakakibara
S
S
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000138:chemical synthesis*; D002470:Cell Survival; D002851:Chromatography, High Pressure Liquid; D016207:Cytokines / Q000138:chemical synthesis*; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D000078792:Midkine; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D012996:Solutions
D002352:Carrier Proteins; D016207:Cytokines; D004220:Disulfides; D012996:Solutions; D000078792:Midkine