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Substrate-baited nanoparticles: a catch and release strategy for enzyme recognition and harvesting.

ABSTRACT: The isolation of a single type of protein from a complex mixture is vital for the characterization of the function, structure, and interactions of the protein of interest and is typically the most laborious aspect of the protein purification process. In this work, a model system is utilized to show the efficacy of synthesizing a "baited" nanoparticle to capture and recycle enzymes (proteins that catalyze chemical reactions) from crude cell lysate. Enzyme trapping and recycling is illustrated with the carbazole 1,9a-dioxygenase (CARDO) system, an enzyme important in bioremediation and natural product synthesis. The enzymes are baited with azide-modified carbazolyl moieties attached to poly(propargyl acrylate) nanoparticles through a click transformation. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicates the single-step procedure to immobilize the enzymes on the particles is capable of significantly concentrating the protein from raw lysate and sequestering all required components of the protein to maintain bioactivity. These results establish a universal model applicable to concentrating and extracting known substrate-protein pairs, but it can be an invaluable tool in recognizing unknown protein-ligand affinities.

SUBMITTER: Daniele MA

PROVIDER: S-EPMC3516911 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Substrate-baited nanoparticles: a catch and release strategy for enzyme recognition and harvesting.

Daniele Michael A MA Bandera Yuriy P YP Sharma Deepti D Rungta Parul P Roeder Ryan R Sehorn Michael G MG Foulger Stephen H SH

Small (Weinheim an der Bergstrasse, Germany) 20120424 13

The isolation of a single type of protein from a complex mixture is vital for the characterization of the function, structure, and interactions of the protein of interest and is typically the most laborious aspect of the protein purification process. In this work, a model system is utilized to show the efficacy of synthesizing a "baited" nanoparticle to capture and recycle enzymes (proteins that catalyze chemical reactions) from crude cell lysate. Enzyme trapping and recycling is illustrated wit ...[more]

PMID: 22532510

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Project description:BackgroundWhen seeking a human for a blood meal, mosquitoes use several cues to detect and find their hosts. From this knowledge, counter-flow odour-baited traps have been developed that use a combination of CO2, human-mimicking odour, visual cues and circulating airflow to attract and capture mosquitoes. Initially developed for monitoring, these traps are now also being considered as promising vector control tools. The traps are attractive to host-seeking mosquitoes, but their capture efficiency is low. It has been hypothesized that the lack of short-range host cues, such as heat and increased local humidity, often prevent mosquitoes from getting close enough to get caught; this lack might even trigger avoidance manoeuvres near the capture region.MethodsThis study investigated how close-range host cues affect the flight behaviour of Anopheles female malaria mosquitoes around odour-baited traps, and how this affects trap capture performance. For this, a novel counter-flow odour-baited trap was developed, the M-Tego. In addition to the usual CO2 and odour-blend, this trap can provide the short-range host cues, heat and humidity. Systematically adding or removing these two cues tested how this affected the trap capture percentages and flight behaviour. First, capture percentages of the M-Tego with and without short-range host cues to the BG-Suna trap were compared, in both laboratory and semi-field testing. Then, machine-vision techniques were used to track the three-dimensional flight movements of mosquitoes around the M-Tego.ResultsWith heat and humidity present, the M-Tego captured significantly more mosquitoes as capture percentages almost doubled. Comparing the flight behaviour around the M-Tego with variable close-range host cues showed that when these cues were present, flying mosquitoes were more attracted to the trap and spent more time there. In addition, the M-Tego was found to have a better capture mechanism than the BG-Suna, most likely because it does not elicit previously observed upward avoiding manoeuvres.ConclusionsResults suggest that adding heat and humidity to an odour-baited trap lures more mosquitoes close to the trap and retains them there longer, resulting in higher capture performance. These findings support the development of control tools for fighting mosquito-borne diseases such as malaria.

Dataset Information

Substrate-baited nanoparticles: a catch and release strategy for enzyme recognition and harvesting.

Publications

Substrate-baited nanoparticles: a catch and release strategy for enzyme recognition and harvesting.

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