Selection And Study Of Cd～(2+)-binding Peptides From Phage Random Peptide Library

Heavy metal pollutions have been a world wide problems all the time. Bioremediation opened an efficient and ecological way for the disposal of heavy metals. One of the key areas is the better utilization of the mechanisms of the strong affinity of metal-binding proteins or peptides for heavy metals in the bioremediation of heavy metals. The rapid development of surface display technology offered an effective and great chance in this area.At present, the most difficult thing is how to obtain the metal binding peptides with high speciality and high affinity and understand the mechanisms of the interactions between heavy metals and metal-binding peptides.The purpose and main contents of this study were to obtain some polypeptides with high affinity for Cd²⁺ by using the phage random dodecapeptide library selection and the immobilized metal affinity chromatography(IMAC);to analyse the characteristics of the obtained peptide sequences by bioinformatics and chemical anlysis;to detect if the metal-binding peptides had effects on the detoxification of heavy metal toxicity;to design and prepare the oligonucleotides and empty plasmids. for the further construction of recombinant bacteria in the hope of the bioremediation of heavy metal pollutions.At first, the Cd²⁺ metal binding peptides were selected and enriched by the imidazole(ID)/glycine(Gly)buffer elution from the filamentous phage dodecapeptide library. After amplification of the eluted phages and the preparation of phage single-strand DNA, ten phage clones were randomly picked up and sequenced. The homology of the Cd²⁺-binding peptides were blasted by GeneBank Blast(http://www. ncbi. nlm. nih. gov/ blast/). The multiple sequence blast of the Cd²⁺ and Ni²⁺ sequences selected by our group and the collected Cd-binding peptides from references were tested by Clustal X 1.81. After the amplification, purification and titering of the monoclonal phages, the relative affinity of these phages for the heavy metal ions(Cu²⁺,Ni²⁺,Co²⁺,Zn²⁺,Cd²⁺,Cr³⁺) chelated resins were determined. After the amplification, infection of ER2738 and plaque inculbation, E. coli paper-ring inhibition experiments were used to determine the detoxification abilities of Cd²⁺-binding peptides displayed phages for heavy metal ions(Cu²⁺,Ni²⁺,Co²⁺,Zn²⁺,Cd²⁺,Cr³⁺) treated E. coli. In addition, by using the DNA recombination technology, the oligonucleotides containing the restriction sites and X-press tag were designed and synthesized, as well as the preparation of the empty plasmid pLBB9 which was used as display vector for the display of the metal binding peptides on the surface of the host cell of E. coli pop6510.The results of the study were as follows: A group of peptides displayed on the surface of phages with different affinities for Cd²⁺ were obtained by the direct selection from the phage random dodecapeptide library with IMAC and phage display technology. Amino acid sequecnce analysis showed they were rich in histidines and having few glycin and no cysteines in the selected peptides. The selected Cd²⁺-binding peptides displayed phages also had affinities for other heavy metal ions(Cu²⁺,Ni²⁺,Co²⁺,Zn²⁺ and Cr³⁺) by the analysis of the affinities of obtained phage clones for different IMACs. They have similar affinities for Cd²⁺ and Cr³⁺, however were more lower than Co²⁺,Ni²⁺,Cu²⁺ and Zn²⁺, and the affinities of peptide No. 1(3) (KSLSRHDHIHHH) were higher than peptide No.2(IFMESLPRLGMH). E. coli paper-ring inhibition experiments showed that Cd²⁺-binding peptides displayed phages had detoxification effects for various heavy metals including Cd²⁺, Cu²⁺, Co²⁺, Ni²⁺, Zn²⁺ and Cr³⁺, the detoxification for Cr³⁺ was the highest while Cd²⁺ was the lowest. The two Cd²⁺-binding peptides had no obvious difference in the detoxification of different metal ions. The difference in the affinities and detoxifications of Cd²⁺-binding peptides for various metals might be explained by the outer electron structure of the metal ions, the expression of Cd²⁺-binding peptides , the metal toxicity and the concentration of the heavy metals. No obvious difference in the detoxification abilities of the two Cd²⁺-binding peptides might be related to the identical function of peptides containing one histidine or multi-histidine as metal chelators. Design of peptide No.1(3) (KSLSRHDHIHHH),identification of the synthesized olionucleotides, as well as the empty plasmids would benefit the construction of recombinant bacteria for the bioremediation of the heavy metal pollution.