In silico design and construction of metal-binding hybrid proteins for specific removal of cadmium based on CS3 pili display on the surface of Escherichia coli

摘要

In this study, through a combination of bioinformatics and genetic engineering procedures, high-affinity metal-binding peptides were designed and expressed on the surface of Escherichia coli for selective Cd~(2+) adsorption. Putative cadmium-binding motifs were identified by searches against the Prosite database and permissive sites in the major subunit (CstH) of the enterotoxigenic E. coli pili were predicted based on the data derived from modeling of 3D structures, secondary structure prediction and assignment, inspection of protein hydropathy and exposed regions, and also protein interaction sites. The metal-binding motifs were inserted into one permissive site of the CstH (amino acid 38) with the aid of the SOEing PCR technique. The capacity and selectivity of the recombinant bacteria displaying hybrid pili to adsorb cadmium were evaluated with the atomic absorption procedure. The levels of Cd~(2+) accumulation in the recombinant E. coli strains were 13.9- and 11.33-fold higher than those in the control strain. Cd~(2+) was selectively absorbed from a solution containing equal concentrations of four metals, resulting in more than 90% of the total adsorbed metals being Cd~(2+), showing a relatively high affinity for Cd~(2+) over other coexisting metal ions.