Screening And Characterization Of Small Peptide Ligands Of TrkA Through Yeast Two-hybrid System

Nerve Growth factor is a member of the superfamily of neutrophic factors that control the development and survival of certain neuronal populations both in the peripheral and in the central nervous system. TrkA, the high affinity NGF receptor, contains an intracellular domain with tyrosine kinase activity and an extracellular domain with three tandem leucine-rich motifs flanked by two cysteine clusters in their amino termini and two immunoglobulin-like domains in the more membrane-proximal region. NGF binding to TrkA receptors results in receptor dimerization and kinase activation.Recent evidence supports that not all extracellular subdomains are responsible for receptor activation. Structure based drug design for neurotrophic agonists with small molecular weight relies on knowledge of the interaction of neurotrophin with their receptors. However, this strategy lacks the ability of high-throughput screening.As the random screening approach requires a target molecule, it is essential to identify the subdomain of TrkA responsible for NGF binding and receptor activation. In present study, the recombinant subdomains of TrkA were expressed and purified. PC 12 cells were used to evaluate their ability to inhibit the neurite outgrowth induced by NGF. The Random Peptide library was screened by using the defined subdomain as a bait in yeast two-hybrid system to obtain small peptides interacting with TrkA receptor.The main results of our research are as follows:1. To obtain purified recombinant TrkA extracellular domain proteins and study their biological activity, the cDNA encoding TrkA extracellular domains (CLC, Igl and Ig2) were amplified by PCR and inserted into the expression vector pET-28a(+) containing T7 promoter and transformed into E.coli BL21(DE3). The SDS-PAGE analysis revealed that the TrkA extracellular domains proteins were highly expressed and accumulated up to above 30% of the total bacterial proteins after IPTG induction. The expressed proteins were purified by Ni2+ chelation affinity chromatography. The recombinant proteins were prepared with the purity more than 95%.2. The functional of the purified TrkA extracellular domain proteins were tested using PC-12 cells. These cells express TrkA and display a neuronal phenotype with neurite extensions after stimulation with NGF. Our results showed that only Ig2 , but not CLC or Ig1, was able to inhibit neurite outgrowth of PC-12 cells. It suggests that the second Ig-like domain of the TrkA receptors is critical for NGF binding and receptor activation.3. The yeast two-hybrid system has been broadly used to identify protein partners. In recent years, protein-peptide interactions have also been analyzed with this system. In our study, the IgII subdomain of TrkA extracellular domain was fused to pGBKT7 as a bait to screen Yeast Random Peptide Library which contains 1 × 107 independent random peptide clones. 328 His+ positive colonies were streaked SD/-Ade/-his/-leu/-trp plates, and 58 Ade+ positive colonies were obtained. 10 colonies were identified finally with colony-lift filter assays forβ -galactosidase activity.4.The plasmids for these 10 positive colonies were cotransformed respectively withpGBKT7-IgII into a yeast reporter strain SFY526 and the interactions between them were evaluated by using liquid 0 -galactosidase activity assay. Peptide P309-1, P309-3, P309-6 and P309-9 produced the strong interaction with IgII of TrkA while P309-2, P309-4, P309-5, P309-7, P309-8 and P309-10 showed weak interaction. We got six peptides from the results of DNA sequencing at last. Among them, P309-1 and P309-6 are 6-residue peptides, P309-3 is 12 -residue peptides, P309-2, P309-7 and P309-8 are 16 -residue peptides.