Tunable biopolymers were synthesized for the removal of heavy metals from dilute waste streams. Protein−protein interaction was tailored specifically into tunable, metal-binding biopolymers using elastin-like polypeptides composed of either one or two hexahistidine clusters. These tunable biopolymers retained the functionality of the elastin domain, undergoing a reversible phase transition above the transition temperature. Aggregation could be tuned within a wide range of temperatures by controlling the chain length of the biopolymers. The presence of the histidine clusters enabled Cd2+ to bind strongly to the biopolymers. Recovery of biopolymer−Cd2+ complexes was easily achieved by triggering aggregation either by raising temperature or by salt addition. Regenerated biopolymers could be reloaded with Cd2+ and reused for repeated cycles with similar efficiency. This system is very flexible as both domains can be engineered to respond to various phase transition conditions and to provide various levels of metal-binding selectivity. The ability to modulate the properties of the biopolymers simply by tuning process conditions should open up new opportunities for the separation and recovery of other environmental contaminants.

The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5-and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd 2؉ or Zn 2؉ accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.Arsenic (As) is an extremely toxic metalloid that adversely affects human health. Both highly toxic trivalent arsenite [As(III)] and less-toxic pentavalent arsenate [As(V)] have been associated with increased risk of skin, kidney, lung, and bladder cancer (12). The toxicity of arsenic is attributed to the substitution of As(V) for phosphate, affinity of As(III) for protein thiol groups, and protein-DNA and DNA-DNA crosslinking (20). Arsenic enters the water supply primarily from geochemical sources, such as the mining of arsenopyrite gold ores (16), which constitute about one-third of world gold reserves. Additional contaminations arise from anthropomorphic sources such as arsenical-containing herbicides, pesticides, and the widely used wood preservative chromated copper arsenic (17, 21). Untreated, highly toxic arsenic effluent has been disposed of in rivers and ended up in groundwater. An estimated 20 million Americans consume water containing arsenic at levels presenting a potentially fatal cancer risk (9). Worldwide, 57 million people have been exposed to arsenic through contaminated wells in Bangladesh (18). These incidents again serve as a reminder of the toxic consequences of arsenic mobilization and the needs for efficient removal of arsenic in aquatic systems (19).Because of the toxicity, the regulatory limit on arsenic in the United States is currently set at 10 ppb. There are a variety of methods currently available for removal of arsenic from contaminated water. Conventional technologies, such as coagulation, do not discriminate between arsenic and other elements and involve alteration of the water chemistry and addition of other chemicals (7). Current technologies, such as activated alumina sorption, polymeric anion exchange, and polymeric ligand exchange (6), are more effective for As(V) than As(III), and most commonly used methods require pri...

A nonmucoid clinical isolate of Pseudomonas aeruginosa, strain 808, elaborated ATP-dependent and ATP-independent types of cytotoxic factors in the growth medium. These cytotoxic factors, active against macrophages, were secreted during the exponential phase of growth in a complex medium. Commensurate with the appearance of the cytotoxic activities in the cell-free growth medium, several ATP-utilizing enzymic activities, such as adenylate kinase, nucleoside diphosphate kinase and 5'-nucleotidase (ATPase and/or phosphatase), were detected in the medium. These ATP-utilizing enzymes are believed to convert external ATP, presumably effluxed from macrophages, to various adenine nucleotides, which then activate purinergic receptors such as P2Z, leading to enhanced macrophage cell death. Pretreatment of macrophages with periodate-oxidized ATP (oATP), which is an irreversible inhibitor of P2Z receptor activation, prevented subsequent ATP-induced macrophage cell death. A second type of cytotoxic factor(s) operated in an ATP-independent manner such that it triggered activation of apoptotic processes in macrophages, leading to proteolytic conversion of procaspase-3 to active caspase-3. This cytotoxic factor(s) did not appear to act on procaspase-3 present in macrophage cytosolic extracts. Intact macrophages, when exposed to the cytotoxic factor(s) for 6-16 h, underwent apoptosis and demonstrated the presence of active caspase-3 in their cytosolic extracts. Interestingly, two redox proteins, azurin and cytochrome c 551 , were detected in the cytotoxic preparation. When cell-line-derived or peritoneal macrophages or mast cells were incubated overnight with Q-Sepharose column flow-through fraction or with a mixture of azurin and cytochrome c 551 , they underwent extensive cell death due to induction of apoptosis.

Leishmania amazonensis was found to release nucleoside diphosphate kinase (NdK) -a stable enzyme capable of decreasing extracellular ATP. The release of this enzyme from Leishmania results in its progressive accumulation extracellularly as they replicate, peaking at the stationary phase in vitro. The released NdK is immunoprecipitable and constitutes ~40% of its total activities and proteins. The retention of a known cytosolic protein by wild type cells and a fluorescent protein by DsRed transfectants at stationary phase, which release NdK, indicates that this is a spontaneous event, independent of inadvertent cytolysis. Recombinant products of Leishmania NdK prepared were enzymatically and immunologically active. Both recombinant and native Leishmania NdK utilized ATP to produce expected nucleoside triphosphates in the presence of nucleoside diphosphates in excess. Both native and recombinant Leishmania NdK were also found to prevent ATP-induced cytolysis of J774 macrophages in vitro, as determined by assays for lactate dehydrogenase release from these cells and for their mitochondrial membrane potential changes. The results obtained thus suggest that Leishmania NdK not only serves its normal house-keeping and other important functions true to all cells, but also prevents ATP-mediated lysis of macrophages, thereby preserving the integrity of the host cells to the benefit of the parasite.