| Heavy metal pollution have already been a worldwide problem. Bioremediation would be a possible way for the well control of environmental pollution and the realization of the sustainable development of both economic and social prosperity, among which the technology of microbial engineering based on biological library and cell surface display caught the interests of many researchers as the result of the many shortcomings of traditional microbial methods. Recent studies showed that the engineering microbes displaying high affinity metal binding peptides would be of great potential in the bioremediation of heavy metals especially the control of low content but high fluent heavy metal pollution.At present the metal binding peptides derived mainly from four sources, (1) 6×His and its derivatives; (2) metallothioneins, metallothionein-like protein, phytochelatin and metal-resistance regulated proteins; (3) mimics of natural metal-containing enzymes or metal transport proteins , or the motifs of metal containing peptides; (4) metal-containing peptides selected from peptide libraries, the major hosts of microbe used for the remediation of heavy metals were predominantly concentrated on bacteria(including G- and G+). Regarding the great potential of phage peptide library and the high capacity of bio-sorption of yeast for heavy metals in addition to its characteristics of generally regarded as safe(GRAS), the work of selection of metal binding peptide and the display of yeast cell surface were proceeded in the following aspects:At first the binding characteristics of filamentous phage peptide library for heavy metal ions (Co2+ and Ni2+) including P/N value(phage tilter of the imidazole (ID) elutes from the biopanning of the immobilized metal affinity chromatography (IMAC) resins divided by that of the resins deprived of metals), relative enrichment and phage clones were studied by IMAC and phage display techniques, phages containing metal binding peptides were enriched and obtained through regional selection and gradient elution by several rounds of biopanning of phage randomdodecapeptide library, the results were as follows: the P/N value of the eluted phages increased with the increasing cycling of biopanning, the number of phage clones reached the height at the ID concentration of 100 mmol/L no matter what regions (20-100;100-200;200-400 mmol/L ID) and different ID elute concentrations (100; 200; 400 mmol/L) ,the phages harvested from different regions showed relative enrichment at different elute concentrations when they were eluted with three kind of ID concentrations, moreover, this kind of relative enrichment was related to the trends of P/N value.On the basis of above work the cobalt cation chelating resins were biopanned directly with elute concentration of 200 mmol/L ID, after five rounds of selection, the P/N value of the eluted phages increased from 1.4 to 140, 19 phage single clones randomly picked up from the 3,4 and 5 rounds of selection and the previous study were assayed by DNA . sequencing, 9 different sequences were obtained and their relevant compositions of amino acids were determined, no consensus sequence was found and they showed no homology to the known metal containing enzymes or their domains by blasting in Genebank (http://www.ncbi.nlm.nih.gov/blast/). The correlated phage clones were amplified and purified and then again assayed for their relative affinity for Co2+ and Ni2+ metal resins by P/N value, phages with variant metal binding affinities were obtained and among which the P/N value of one phage clone reached the highest to 1970 and 7200 for Co2+ and Ni2+ respectively. Considering the high ratio of histidine in the peptides and convinced proof of the high affinity of 6xHis for heavy metals, recombinant phage M13 in the structure construction of random dodecapeptide library with the insertion of 12xHis from M13RF were constructed. Restriction analysis and Western blotting confirmed the correct construction and display of 12xHis of the recombinant phage, relative affinity assay really showed its great affinity for metals with high P/N values for cobalt and nickel metal cation chelating resins, for this reason, four sequences representing the different affinities for heavy metals named 7H, 5H, Rd and 12H(the relevant recombinant phage clones were named as Ph/7H, Ph/5H, Ph/Rd and Ph/12H respectively)were selected, Western blotting of the whole phage protein showed that Ph/12H, Ph/7H and Ph/5H were reactive with 6xHis monoclonal antibody and Ph/12H was the most strongest among the three recombinant phages.The successful obtainment of high affinity metal binding peptides offered valuable sequences for display and the specific detection of peptides riched in histidines provided an easy and efficient method for the detection of the targeted peptides on the surface of the yeast cells, by the introduction of restriction sites and the synthesis of the oligonucleotides, the four specific sequences 12H, 7H. 5H and Rd were fused to the C-terminus of Aga2p of the a-agglutinin receptor of yeast cell wall by recombinant DNA technology. After the selection under the auxotrophic factor TRP, positive yeast clones named as E/12H, E/7H, E/5H, E/Rd respectively were obtained. Restriction analysis, PAGE DNA electrophoresis and DNA sequencing confirmed the correct construction of the positive clones. Taking the host yeast (named as E) and the recombinant yeast with the blank plasmid containing 6xHis(named as E/6H) as controls, expressions of fused peptides were assayed by solid phase yeast ELISA with or without disposal ofDTT. The results showed the sufficient display of the fused peptides of the recombinant yeasts E/12H, E/7H, E/5H and E/6H under the induction of p-galactose at temperature of 23—25 "C.With the success of the display of the peptides on the surface of recombinant yeasts the endurance and bio-sorption of the displayed yeasts for heavy metals were studied, the two recombinant yeasts E/7H and E/Rd showed the same in their endurance characteristics for the three heavy metal ions(Cd2+ Ni2+ and Co2+) even though there were dominant difference in the endurance of heavy metal concentrations, It disclosed that there were no dominant impact of heavy metal disposal on the growth character of recombinant yeasts displaying different kind of fused peptides. Experiment of heavy metal disposal showed that with the increment of heavy metal concentrations, there was an increase in the metal bio-sorption of yeasts, at the final concentration of 100(imol/L of Cd2+, after incubation for lhour, E/7H was the most strongest strain for the bio-sorption of Cd2+ among all the detected yeasts with its average cadmium content to 106.8 jj.g/g, similarly, at the concentration of 500(imol/L of Ni2+of the similar conditions, E/7H was also the strongest, furthermore, under the disposal of different concentrations of Ni2+ (5 > 50. 500, 5000|amol/L), there was a great trend that the contents of nickel was much more higher in E/7H than in E/Rd.Conclusions:A group of peptides displayed on the phages with different affinity for heavy metals were obtained by direct and fractional selection from the phage random dodecapeptide library with IMAC and phage display technology. Analysis of the amino acids showed high composition of histidine in the inserted peptides of phages. Recombinant yeasts displaying metal binding peptides showed strong greater bio-sorption of heavy metals as compared with wild type yeast and the yeast displaying the random sequence, moreover, both the phage and yeast displaying 7H sequence showed highest affinity for heavy metals, indicating the possibility of constructing genetically engineered yeast by selecting binding peptides from random peptide library and then displaying on the surface of yeast cells. The research thus opened an efficient way for the bioremediation of heavy metals and radionuclides and also other environmental pollutants.
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