Screening, Synthesis And Quantum Dot Labeling Of Short Peptide Binding Affinity To Insulin Receptor

Insulin is a kind of protein hormone secreted by pancreas islet βcells. It is well knownthat insulin maintains glucose homeostasis by adjusting glucose intake and oxidation andglycogen generation and store. The complex physiological action of insulin depends on itsbinding to insulin receptor. The insulin receptor is an intrinsic disulfide-linked dimer of heterodimericdisulfide-linked proteins of the form (αβ)2. The alpha subunits are entirely extracellular andcontain the ligand-binding domain. For many years, the binding mechanism of insulin with its receptor has always beenmended, however, it is generally thought that insulin has two binding sites. Insulin proximityto one monomer juxtaposes major hydrophobic areas on the insulin B chain and on L1, aswell as charged amino acids with matching side chains on the C2 and L2 regions. Interactionsof insulin with the other monomer are predominantly electrostatic, with no obvioushydrophobic components. The beta subunits contain a transmembrane domain. Their cytoplasmic domains play animportant role in IR signal transduction. Binding with insulin induces itsautophosphorylation and therefore activates its tyrosine kinase activity. The phosphorylatedIR subsequently interacts with the insulin receptor substrates known as cytoplasmatic signalprotein(IRS-1,IRS-2 et al), inducing the phosphorylation of several Tyr residues on the IRS.Interction between phosphorylated IRS with SH2 protein(eg. PI3' kinase) mediates the signal.Although much is know about the IR in terms of its mode of action and signaling pathways,there is a paucity of information regarding the molecular architecture of the insulin bindingsite, the contact sites for insulin binding and the mechanoisms by which insulin induces itsbiological effects. People have been devoted in researching the hot spots on IR and on insulin binding sites.They hope to clear these doubts and obtain the minimum while act as full binding actionfragments. Insulin can not be taken orally or by nasal cavity, otherwise, will be decomposed.Mainline is the only way for diabetics, but it has its drawback that the high concentrationabove physiological one makes insulin polymerization and impede its effect. Thereforeseeking and designing small analogs for making oral surrogate of insulin is of greatimportance. It is a right way to screen in a phage-display random peptide library.Our group takes the Chinese hamster ovary cells overexpressing insulin receptors(CHO-IR) astargets to sceen CHO-IR binding peptides in a phage display hexapeptide library. After fourround of panning, we obtain six ELISA positive recombined clones whose Kds all range innanomolar scale(5.911nM～26.899nM).Considering the Kd values as main indexescombinding with the yield ratio would lead to more preferable results. The amino acidsequences of these peptides display distinct conservation with each other. Five of those peptides are synthesized by automated peptide synthesizer and manual way.And the D-biotins are coupled to the N-terminal in order to get both biotin and non-biotinpeptides. Determined by MS and biotin-avidin binding reaction, this conjugating methoddon't affect biotin binding with avidin. What's more, by this way the trifling purificationusing NHS-biotin to biotinylate peptides are avoided and D-biotin are much cheaper thanNHS-biotin. The Kd values of the five peptides are measured by ELISA and PRISM4.0software which range from 5.870μM~128.6μM and belong to specific affinity scope. Compare the Kd values of the peptide with those of the phage clones, differences rangein 102-104 exist which should owe to the enlarged sigals in the former when determing withantibody and then the IgG. In addition, several amino acids nearby the displayed peptide onthe phage epitope may donate to these binding. However, this result would be a goodreference for later screening from phage display random peptide library. To understand more fully the interaction of peptides with their receptor, we usecompeting ELISA to assay the interaction of mixed peptides among which onl...