Selection And Interaction Of Ni～(2+) Metal-Binding Peptides

Heavy metal pollution has in a variety of environments been a severe threat to the human's health which is regarded as a world-wide problem all the time. Many heavy metals, such as Hg,Cd,Pb and Cr, usually are toxic for many organism.Even some organisms' essential trace elements, such as copper, zinc and nickle also produce obvious toxicity effect against the living creature at higher concentration.Heavy metals can bind to proteins and reduce their activities or break their structure, replace some essential trace elements and thus cause the lack of nourishment, even to induce and create the formation of the free group or live oxygen.There are many metal-binding peptides with heavy metals affinities that were found in plants,microorganisms and animals. There are a lot of reports about metal-binding polypeptides(proteins)which mainly concentrated on the nature polypeptides(proteins), such as Phytochelatins(PCs) and Metallothioneins (MTs), however there are still few reports about the short and new metal-binding polypeptides which are of special merits and have great values in the heavy metal bioremediationon.In order to find high affinity Ni²⁺ binding ppetides, phage display technique was utilized.The phage library was amplified and later the quantified phages were added into Ni²⁺-resins, washed with TBST buffer and at last eluted by imidazole (ID) solutions. The procedure was repeated for six times, the 12-peptides with affinity for Ni²⁺ were successfully selected.Phage clones were selected randomly and the DNA sequence was determined for each clone. Four different animo acid sequences were obtained which were found containing four to five histidine residues.In this report, the affinities of the metal binding peptides acquired from random peptide phage library against different metal chelating resins and the tolerance and the detoxification of the phage displaying metal peptide against heavy metal were assayed. Phage display technique, bacteria repression experiment and the physical observation of the binding of phages to heavy metal chelated resins under microscope were used to measure the affinity and/or speciality of heavy metal chelating resins against the phage clones which displayed Ni²⁺ metal-binding polypeptide. Results were as follows: the prepared metal- chelated resins could be used to detect the affinity of Ni²⁺ metal-binding polypeptides; the obtained meta-binding peptides had the similar effects to those of other heavy metals; Cu²⁺ Ni²⁺ Co²⁺ and Zn²⁺ had higher affinities for Ni²⁺ metal-binding polypeptides than those of Cd²⁺ Cr²⁺; sequences were rich in the content of His amino acid, indicating the content of His amino acid in polypeptide playing an important role in the binding of heavy metals phages displaying metal-binding polypeptides increased their duration and detoxification of heavy metal in a repression assay; at the same time, the resin's viscosity was affected after the metal-binding polypeptide displayed phages mixing with the metal chelating. It is of great value to acquire effective and special metal peptides resins both for the bioremediation of the heavy metals and the study of the interactions between heavy metals and metal-inding peptides.In this report, the construction of bacterial-displayed random peptide library on metal binding protein was also approached preliminarily.At present ,considering that we didn't found metal binding peptides which were selected from phage display technology contain important amino acids such as Cys.however,from metal binding protein(polypeptide) such as MTs , Zinc Fingers and so on, we have already understood that Cys plays an important role in binding metal .In this thesis ,we used the scaffold of Cys2His2 zinc finger polypeptide, and completed the design and synthesis of the oligonucleotide random peptide library ,as well as the preparation of the empty vector of bacterial plasmid pLBB9, intending to insert the fragment of Zinc Finger into the plasmid to construct the recombinant plasmid which displayed on the surface of bacteria. We hope to obtain Cys—His containging binding-peptide sequences by screening of the constructed random peptide library, which offering the theoretical basis for the better understanding of the mechanisms of interaction between polypeptide in Zinc Finger Proteins(peptides) and heavy metals with high affinity, as well as for the study of interaction between metal binding peptide and metal ions.