Identification Of Peptides That Bind To Pisum Sativum Agglutinin (PSA)

[Objective]Using phage display, we can fix on antigenic determinants and find the characteristic sequences that the proteolytic enzymes act and the site proteins interacting together. Now, we also obtain mimic of some non-peptide molecule (saccharides) by phage display. The interaction of carbohydrate and proteins is the pathology basic of a lot of diseases. If we can filtrate the mimeticsc of the saccharide that participate in certain pathologic processes from a phage display random peptide library, we can find a new way to explore the drugs for these diseases.[methods]1. PSA can specially bind with glucose, so we purified PSA by affinity chromatography with sephedax-G50. Then we detected the activity of cruor of the product and identify it by SDS-PAGE electrophoresis.2. We screened a phage-displayed random hexapeptide library with Pisumsativum agglutinin (PSA) .3. The phages can be sorted into three groups. We selected 1² clones from each group, and use α-Met-D-mannoside to inhibit PSA binding to these phages.4. We selectively synthesized three peptides(ARMWSF, RYDYSY, LRLRQL), and use different concentration of these peptides to inhibit PSA and ConA binding to the HRP.[Results]1. The product can obviously mass the blood corpuscle, and the result of SDS-PAGE indicated that the product is pure PSA.2. The enrichment occurred obviously after three rounds of screening. The insert sequences of amino acids, displayed on 22 phage DNAs isolated from the third round of screening, were divided into three groups.3. We found the phages that contain different peptide sequences have different affinity with the a-Met-D-mannoside.4. LRLRQL was not dissolved in water. ARMWSF and RYDYSY inhibited binding of PSA to HRP, but failed to inhibit binding ConA to HRP.[Conclusion]The above results suggest that we can successfully obtain the peptides that specially binding to PSA by phage display. The binding of the PSA and the phages that contain the peptides can be inhibited by a-Met-D-mannoside. There are maybe two reasons: â‘ The peptide compete the site that a-Met-D-mannoside bind to PSA. â‘¡The structure of the binding site of PSA bind to the a-Met-D-mannoside has changed after the peptides bind to PSA.According to IC₅₀, we selectively synthesized three peptides( ARMWSF, RYDYSY, LRLRQL), and use different concentration of these peptides to inhibit PSA and ConA binding to the HRP. We found that LRLQL is not dissolved inwater, this remind us that hydrophobic property forecast of the peptides is necessary before we synthesize them. The binding site of ARMWSF and RYDYSY is independent of the a-Met-D-mannoside binding site. Maybe the binding of these two peptides to PSA influence a-Met-D-mannoside binding to PSA.