Cadmium detection by a thermally responsive elastin copolymer with metal-binding functionality.

Heavy metals are of great concern to environmental safety because of their adverse effects on the environment and human health, even at very low levels. In particular, cadmium and several cadmium-containing compounds are carcinogens and induce many types of cancer. Biological extracts of cadmium have been given greater attention recently because they are considered to be environmentally benign and economically acceptable. Among promising candidates, one emerging technology is the use of tunable, metal-binding biopolymers based on elastin-like polypeptides (ELPs). An ELP consists of the repeating pentapeptide of specific amino acids, Val-Pro-Gly-Xaa-Gly (where the "guest residue" Xaa is any amino except proline) that undergoes a reversible phase transition at a specific temperature (transition temperature, Tt). However, the ELP itself is relatively non-selective. A biopolymer with metal-binding domains that have stronger affinity, capacity, and selectivity would have distinct advantages. We investigated the use of a new generation of ELP biopolymers, EC18-ELP containing synthetic phytochelatin (EC), which is a metal-binding protein with a repetitive motif (Glu-Cys)nGly, as the metal-binding domain. In this study, an EC18-ELP fusion protein was expressed in Escherichia coli and the metal binding ability of EC to cadmium was examined quantitatively. In addition, transition temperature variation was analyzed when the fusion protein bound to cadmium.

A new cloning strategy for generating multiple repeats of a repetitive polypeptide based on non-template PCR.

A new cloning method for generating multiple repeats of amino acids is described which can be used as biomaterials, protein polymers and biomedical applications. Although several traditional methods for cloning multiple repeats are still exploited, these are laborious and complicated because they must go through several consecutive cloning steps. To solve these problems, synthetic gene libraries encoding repetitive patterns were constructed by using non-template PCR. As a result, a 'length library' with fourteen different ELP repeating genes was constructed and expressed in a cell-free protein synthesis system. These results showed our novel cloning method is efficient, and has the potential capacity for synthesizing repetitive genes by PCR to be cloned in any commercial expression vectors.