For the purpose of total synthesis of human insulin, enzymatic method was applied for the fragment condensation of A or B chain, together with the preparation of single chain insulin by coupling of fragments A and B chains.
Eight insulin fragments were synthesized using a peptide synthesizer (Shimadzu model PSSM-8). For coupling, Staphylococcus aureus V8 protease (abbrev.SAP), trypsin (abbrev.T) and lysyl endopeptidase (abbrev.L) were used. The reaction was performed in the persence of high concentration of organic cosolvent (40% dimethylformamide). The reaction mixture contained 2 mM amine component and 10 mM carboxyl component, pH 6-7, which was kept for 1-3 days at room temperature. The other experimental conditions are described below. The coupling yield was determined using high performance liquid chromatography (HPLC). The result indicated that the peak of amine component disappeared and a new peak appeared in HPLC in all the coupling reactions below. Amino acid analysis of the respective new peaks in HPLC indicated that the new peaks were the coupling products as had been expected.
Abbrev. : A ; insulin A chain, Cys6-Cys11 (S-S bridge), Cys7-Acm, Cys20-Acm.B ; insulin B chain, Cys7-Acm, Cys19-Acm.Acm, acetoamidemethy1..
The numbers in parentheses are the amino acid ones in the respective sequences.
B(1-13) B(14-22) SAP B(1-22)
B(1-22) B(23-30) T B(1-30)
A(1-21) B(1-29) L B(1-29)-A(1-21)