Solid Phase Synthesis And Separation Application Of Peptide Affinity Ligands For Insulin

A kind of resin (PGE for short) prepared by radical suspension polymerization (GMA used as monomer and EDMA used as crosslinking agent) is used to synthesize peptide affinity ligand for insulin in the method of Fmoc SPPS (Solid Phase Peptide Synthesis) in this dissertation. And the ligand is studied in the affinity absorption experiment. Firstly, we synthesis heptapeptide ligand (HWWWPAS) with high affinity specialty to insulin,which is screened from phage display peptide library. In the experiment, we find that Pro in the sequence can cause intramolecular aminolysis at the dipeptide level and lead to the formation of diketopiperazines (DKP) as a serious side reaction, which not only leads to a lower overall yield, but also to the presence in the reaction crude of several deletion peptides. Besides this, when some amino acid with small bulk side chain appear around the C-terminal, the result will be poor. So, we delete the Pro in the sequence and substitute Ala with Glu, and then hexapeptide HWWWES is successfully synthesized. We immobilize it on Sepharose to study its absorption performance. The result indicate that hexapeptide HWWWES has the similar property with heptapeptide HWWWPAS as ligand for insulin. PGE is prepared and modified with NH3·H2O, and then acts as vector to synthesis affinity ligand PGE-hexapeptide. The product is used to pack the affinity column to carry out absorption experiment using Insulin,Lysozyme,RNase and amylase as target protein. The result shows this kind of affinity medium has higher specialty to insulin compared with hexapeptide immobilized on Sepharose, and can separate mixed proteins even using higher flow speed.