Biomedical subjects

Wilfred Chen

Publications and source records attributed to Wilfred Chen.

49 records · Page 3Linked to original sources

PubMed2003

Thermally triggered purification and immobilization of elastin-OPH fusions.

A bifunctional fusion protein consisting of organophosphorus hydrolase (OPH) and elastin-like polypeptide (ELP) was synthesized for the detoxification of organophosphorus compounds. ELPs undergo a reversible phase transition upon an increase in temperature, forming hydrophobic aggregates. This thermally triggered property of phase transition allows for a simple and rapid means of purifying the fusion protein. Over 1,300-fold purification was achieved after only 2 cycles of inverse phase transition. The purified fusion protein showed identical kinetic properties as the native OPH with only a modest 10% increase in K(m) and a 5% decrease of K(cat). The ability of the ELP domain to form collapsed aggregates also improved long-term stability of the fusion enzyme. Aggregated ELP-OPH retained nearly 100% activity over a span of three weeks. In addition to facilitating purification and stability, the ELP moiety served as a hydrophobic tag for one-step immobilization of the fusion protein onto hydrophobic surfaces. The ELP-OPH was capable of rapidly degrading paraoxon while immobilized. The protein also retained ELP functionality of reversible phase transition thereby allowing for the regeneration of the treated surface. This technology offers a swift and convenient means for purification, immobilization, and regeneration of OPH onto a variety of hydrophobic surfaces by simple environmental triggers.

Aryldialkylphosphatase↗

PubMed2003

Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection.

A novel capacitance biosensor based on synthetic phytochelatins for sensitive detection of heavy metals is described. Synthetic phytochelatin (Glu-Cys)(20)Gly (EC20) fused to the maltose binding domain protein was expressed in Escherichia coli and purified for construction of the biosensor. The new biosensor was able to detect Hg(2+), Cd(2+), Pb(2+), Cu(2+) and Zn(2+) ions in concentration range of 100 fM-10 mM, and the order of sensitivity was S(Zn)>S(Cu)>S(Hg)>>S(Cd) congruent with S(Pb). The biological sensing element of the sensor could be regenerated using EDTA and the storage stability of the biosensor was 15 days.

Biosensing Techniques↗

PubMed2003

Enhanced mercury biosorption by bacterial cells with surface-displayed MerR.

The metalloregulatory protein MerR, which exhibits high affinity and selectivity toward mercury, was exploited for the construction of microbial biosorbents specific for mercury removal. Whole-cell sorbents were constructed with MerR genetically engineered onto the surface of Escherichia coli cells by using an ice nucleation protein anchor. The presence of surface-exposed MerR on the engineered strains enabled sixfold-higher Hg(2+) biosorption than that found in the wild-type JM109 cells. Hg(2+) binding via MerR was very specific, with no observable decline even in the presence of 100-fold excess Cd(2+) and Zn(2+). The Hg(2+) binding property of the whole-cell sorbents was also insensitive to different ionic strengths, pHs, and the presence of metal chelators. Since metalloregulatory proteins are currently available for a wide variety of toxic heavy metals, our results suggest that microbial biosorbents overexpressing metalloregulatory proteins may be used similarly for the cleanup of other important heavy metals.

Adsorption↗

PubMed2002

Cell-Surface display of heterologous proteins: From high-throughput screening to environmental applications.

A variety of expression systems for the display of either short peptides or fully folded proteins on E.coli and, to a lesser extent, on Gram-positive bacteria have been developed. The expression of proteins on the surface of microbial cells has proved extremely important for numerous applications ranging from combinatorial library screening and protein engineering, to whole cell biocatalysts and adsorbants for bioremediation purposes.

Bacterial Proteins↗

PubMed2002

Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation.

Synthetic phytochelatins (ECs) composed of (Glu-Cys)nGly are protein analogs of phytochelatin that exhibit improved metal-binding capacity over metallothioneins (MTs). Expression of EC20 on the surface of E. coli using the Lpp-OmpA anchor resulted in improved bioaccumulation of cadmium and mercury, providing a new method for treating heavy metal contamination. To further improve the whole-cell accumulation of heavy metals, EC20 was expressed on the surface of Moraxella sp., a bacterium known to survive in contaminated environments, using the truncated ice nucleation protein (INPNC) anchor. Production of EC20 was approximately three-fold higher in Moraxella sp. than E. coli. As a consequence, the mercury-binding capacity of the recombinant Moraxella sp. was increased by more than 10-fold. Owing to the very high level of surface expression, the use of Moraxella sp. and INPNC anchor may prove to be useful for the remediation of other environmental contaminants.

Bacterial Outer Membrane Proteins↗

PubMed2002

Specific adhesion to cellulose and hydrolysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase.

A genetically engineered Escherichia coli cell expressing both organophosphorus hydrolase (OPH) and a cellulose-binding domain (CBD) on the cell surface was constructed, enabling the simultaneous hydrolysis of organophosphate nerve agents and immobilization via specific adsorption to cellulose. OPH was displayed on the cell surface by use of the truncated ice nucleation protein (INPNC) fusion system, while the CBD was surface anchored by the Lpp-OmpA fusion system. Production of both INPNC-OPH and Lpp-OmpA-CBD fusion proteins was verified by immunoblotting, and the surface localization of OPH and the CBD was confirmed by immunofluorescence microscopy. Whole-cell immobilization with the surface-anchored CBD was very specific, forming essentially a monolayer of cells on different supports, as shown by electron micrographs. Optimal levels of OPH activity and binding affinity to cellulose supports were achieved by investigating expression under different induction levels. Immobilized cells degraded paraoxon rapidly at an initial rate of 0.65 mM/min/g of cells (dry weight) and retained almost 100% efficiency over a period of 45 days. Owing to its superior degradation capacity and affinity to cellulose, this immobilized-cell system should be an attractive alternative for large-scale detoxification of organophosphate nerve agents.

Bacterial Adhesion↗

PubMed2002

Bacterial cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents.

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.

Amino Acid Substitution↗

PubMedDate not supplied

Heavy metal removal by novel CBD-EC20 sorbents immobilized on cellulose.

Heavy metals are major contributors to pollution of the biosphere, and their efficient removal from contaminated water is required. Biosorption is an emerging technology that has been shown to be effective in removing very low levels of heavy metal from wastewater. Although peptides such as metallothioneins or phytotchelatins are known to immobilize heavy metals, peptide-based biosorbents have not been extensively investigated. In this paper, we describe the construction and expression of bifunctional fusion proteins consisting of synthetic phytochelatin (EC20) linked to a Clostridium-derived cellulose-binding domain (CBD(clos)), enabling purification and immobilization of the fusions onto different cellulose materials in essentially a single step. The immobilized sorbents were shown to be highly effective in removing cadmium at parts per million levels. Repeated removal of cadmium was demonstrated in an immobilized column. The ability to genetically engineer biosorbents with precisely defined properties could provide an attractive strategy for developing high-affinity bioadsorbents suitable for heavy metal removal.

Adsorption↗

PubMedDate not supplied

Cell surface display of organophosphorus hydrolase in Pseudomonas putida using an ice-nucleation protein anchor.

A surface anchor system derived from the ice-nucleation protein (INP) from Pseudomonas syringe was used to localize organophosphorus hydrolase (OPH) onto the surface of Pseudomonas putida KT2440. Cells harboring the shuttle vector pPNCO33 coding for the INP-OPH fusion were capable of targeting OPH onto the cell surface as demonstrated by whole cell ELISA. The whole cell activity of P. putida KT2440 was shown to be 10 times higher than those of previous efforts expressing the same fusion protein in Escherichia coli. The capability of expressing enzymes on the surface of a robust and environmentally benign P. putida KT2440 should open up new avenues for a wide range of applications such as in situ bioremediation.

Aryldialkylphosphatase↗

PubMedDate not supplied

Detection of benzene, toluene, ethyl benzene, and xylenes (BTEX) using toluene dioxygenase-peroxidase coupling reactions.

We have developed a simple, whole-cell bioassay for the detection of bioavailable benzene, toluene, ethyl benzene, and xylenes (BTEX) and similar compounds. A genetically engineered E. coli strain expressing toluene dioxygenase (TDO) and toluene dihydrodiol dehydrogenase (TodD) was constructed, enabling the conversion of BTEX into their respective catechols, which were quickly converted into colored products by a horseradish peroxidase (HRP)-coupled reaction. The intensity of the color formation was correlated to concentrations of the BTEX compounds. Under the optimized conditions, a detection limit (defined as three times the standard deviation of the response obtained from the blank) of 10, 10, 20, and 50 microM was observed for benzene, toluene, ethyl benzene, and xylene, respectively. The bioassay was selective toward BTEX-related compounds with no interference observed with commonly used pesticides, herbicides, and organic solvent. The bioassay was very stable with little change in response over a 10-week period. The excellent stability suggests that the reported bioassay may be suitable for field monitoring of BTEX to identify and track contaminated water and follow the bioremediation progress.

Benzene↗

PubMedDate not supplied

Improved degradation of organophosphorus nerve agents and p-nitrophenol by Pseudomonas putida JS444 with surface-expressed organophosphorus hydrolase.

Pseudomonas putida JS444, isolated from p-nitrophenol (PNP) contaminated waste sites, was genetically engineered to simultaneously degrade organophosphorus pesticides (OP) and PNP. A surface anchor system derived from the ice-nucleation protein (INP) from Pseudomonas syringae was used to target the organophosphorus hydrolase (OPH) onto the surface of Pseudomonas putida JS444, reducing the potential substrate uptake limitation. Engineered cells were capable of targeting OPH onto the cell surface as demonstrated by western blotting, cell fractionation, and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without instability problems associated with the engineered Moraxella sp. The initial hydrolysis rate was 7.90, 3.54, and 1.53 micromol/h/mg dry weight for paraoxon, parathion, and methyl parathion, respectively. The excellent stability in combination with the rapid degradation rate for organophosphates and PNP make this engineered strain an ideal biocatalyst for complete mineralization of organophosphates.

Aryldialkylphosphatase↗

PubMedDate not supplied

Bioremediation of organophosphorus pesticides by surface-expressed carboxylesterase from mosquito on Escherichia coli.

The insecticide resistance-associated esterase, carboxylesterase B1 (CaE B1), from mosquito was used to degrade the organophosphorus compounds. To eradicate the need for enzyme purification and minimize the resistance to mass transport of the substrate and product across the cell membranes, the CaE B1 was displayed on the cell surface of Escherichia coli fused to the C-terminus of the ice nucleation protein (INP). The presence of CaE B1 on the bacterial cell surface was verified by SDS-PAGE, Western blotting analysis, and immunofluorescence microscopy. More than 50% of active CaE B1 is exported across the membrane and anchored onto the cell surface as determined by proteinase accessibility and cell fractionation experiments. In contrast, only a 6% drop in activity for proteinase K-treated cells was detected from E.coli cells containing pET-B1. From the degradation experiment, more than 80% of the malathion was degraded by whole cells containing plasmid pUC-NC-B1. Constitutive expression of CaE B1 on the surface using INPNC resulted in no cell lysis, and the suspended cultures also exhibited good stability. Because of their high biodegradation activity and superior stability, these "live biocatalysts" are promising for detoxification of organophosphorus pesticides.

Animals↗

PubMedDate not supplied

Surface display of organophosphorus hydrolase on Saccharomyces cerevisiae.

The gene encoding organophosphorus hydrolase (OPH) from Flavobacterium species was expressed on the cell surface of Saccharomyces cerevisiae MT8-1 using a glycosylphosphatidylinositol (GPI) anchor linked to the C-terminal region of OPH. Immunofluorescence microscopy confirmed the localization of OPH on the cell surface, and fluorescence intensity measurement of cells revealed that 1.4 x 10(4) molecules of OPH per cell were displayed. Seventy percent of OPH whole-cell activity was detected on the cell surface by protease accessibility assay. The activity of OPH was highly dependent on cell growth conditions. The maximum activity was obtained when cells were grown in a synthetic dextrose medium lacking tryptophan (SD-W) buffered by 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, 200 mM, pH 7.0) at 20 degrees C, and cobalt chloride was added at 0.1 mM. S. cerevisiae MT8-1 displaying OPH which exhibited a higher activity than Escherichia coli displaying OPH using the ice nucleation protein (INP) anchor. The use of S. cerevisiae MT8-1, which has a "generally regarded as safe (GRAS)" status, as a host for the easy expression of the OPH gene provides a new biocatalyst useful for simultaneous detoxification and detection of organophosphorus pesticides.

Aryldialkylphosphatase↗