Use of GFP as a Reporter for the Facile Analysis of Sequence-Specific Proteases

Pehrson, J.C.; Weatherman, Aaron; Markwell, John; Sarath, Gautam; Schwartzbach, Steven D.

doi:10.2144/99271bm04

Cited by 7 publications

(7 citation statements)

References 2 publications

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“…These type of fluorescent substrates have been used in both in vitro and cell based assays (10,28). In a related approach to that which we describe, a fluorescent or phage associated substrate is linked to an immobile support matrix, such as a bead, and enzyme mediated cleavage of a linking substrate molecule results in a change in fluorescence emission properties, or release of bound fluorophores or phages (24,(29)(30)(31). Although fluorescent quenched substrate libraries are a very powerful tool for investigation of enzymatic activity and specificity, potential disadvantages of using fluorophore based assays are the tendency of chromophores to interact with either the active site of the enzyme or the substrate (32), the limitations to short intramolecular distances due to the efficiency of the fluorescence resonance energy transfer (FRET) as a function of the Forster distance (9), and the necessity for many cleavage reactions to occur to get a reasonable signal (33).…”

Section: Discussionmentioning

confidence: 99%

Enzyme-mediated individual nanoparticle release assay

Glass

Dickerson

Schultz

2006

Analytical Biochemistry

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Numerous methods have been developed to measure the presence of macromolecular species in a sample; however, the number of methods that detect functional activity or modulators of that activity is more limited. To address this limitation, an approach was developed that uses the optical detection of nanoparticles as a measure of enzyme activity. Nanoparticles are increasingly being used as biological labels in static binding assays; here, we describe their use in a release assay format, where the enzyme-mediated liberation of individual nanoparticles from a surface is measured. A double-stranded fragment of DNA is used as the initial tether to bind the nanoparticles to a solid surface. The nanoparticle spatial distribution and number are determined using dark-field optical microscopy and digital image capture. Site-specific cleavage of the DNA tether results in nanoparticle release. The methodology and validation of this approach for measuring enzyme-mediated, individual DNA cleavage events, rapidly, with high specificity, and in real-time are described. This approach was used to detect and discriminate between nonmethylated and methylated DNA, and demonstrates a novel platform for high-throughput screening of modulators of enzyme activity.

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Section: Discussionmentioning

confidence: 99%

Enzyme-mediated individual nanoparticle release assay

Glass

Dickerson

Schultz

2006

Analytical Biochemistry

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“…Another fusion protein substrate has also been developed for in vitro analyzing SUMO-specific protease [17]. However, assays of these designed fusion protein substrates are inconvenient, because separation of the cleaved and uncleaved products is required to quantify the cleavage [12,13,18-20]. In addition, the ability of several protein substrates is further limited by their low stability under relatively high temperature or in the presence of denaturant [7,12-14].…”

Section: Backgoundmentioning

confidence: 99%

A new fusion protein platform for quantitatively measuring activity of multiple proteases

et al. 2014

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BackgroundRecombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases.ResultsWe tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract.ConclusionThe results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with sufficient substrate specificity.

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“…However, unlike a short peptide substrate with convenient probes such as a fluorogenic moiety, protease assays for macro-protein substrates heavily rely on either chromatographic separation techniques or SDS-PAGE analysis for the size alteration of the substrates. Recently, an assay using enhanced green fluorescent protein (EGFP) fluorescence to detect cleavage by Thrombin has been reported (18). However, this assay has a major limitation-insolubility of the substrate, which limits its utility.…”

Section: Research Reportmentioning

confidence: 99%

In Vitro Assay for Site-Specific Proteases Using Bead-Attached GFP Substrate

et al. 2001

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Site-specific proteases, which catalyze cleavage of peptide bonds in specific amino acid sequences of target proteins, play important roles in various biological events of many living organisms. In humans, disruption in regulation of these site-specific proteases can lead to pathological consequences. Here, we report a simple in vitro assay for enzymatic activities of site-specific proteases. This assay system employs a protein substrate molecule that is comprised of (i) His-tag binding module, (ii) cleavage sites, and (iii) green fluorescent protein (GFP) detection module. In this study, prostate-specific antigen (PSA) and Thrombin-specific cleavage sites were introduced into the substrate molecules. The overexpressed GFP substrate protein was purified with the aid of Ni++-charged magnetic beads. On cleavage by either PSA or Thrombin, GFP was released from the bound magnetic beads, enabling a direct measurement of the cleaved product by fluorescence. Detection sensitivity, as well as the kinetics of reaction of PSA cleavage with the GFP substrate, was similar or better than commercially available PSA fluorogenic peptide substrate. This bead-attached GFP substrate was also used for an inhibition assay using a competitive inhibitor of Thrombin. In conclusion, this assay offers a simple fluorescent method for monitoring the activity of the site-specific proteases. Furthermore, this system provides flexible means of incorporating varying sizes of flanking sequences adjacent to the cleavage site, which can be essential for studying the regulatory macromolecular interactions between proteases and their substrates.

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Use of GFP as a Reporter for the Facile Analysis of Sequence-Specific Proteases

Cited by 7 publications

References 2 publications

Enzyme-mediated individual nanoparticle release assay

Enzyme-mediated individual nanoparticle release assay

A new fusion protein platform for quantitatively measuring activity of multiple proteases

In Vitro Assay for Site-Specific Proteases Using Bead-Attached GFP Substrate

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