Difference between revisions of "Team:Slovenia/Protease signaling/Reporters"

 
(39 intermediate revisions by 5 users not shown)
Line 7: Line 7:
 
     <script type="text/javascript"
 
     <script type="text/javascript"
 
             src="//2016.igem.org/Team:Slovenia/libraries/semantic-min-js?action=raw&ctype=text/javascript"></script>
 
             src="//2016.igem.org/Team:Slovenia/libraries/semantic-min-js?action=raw&ctype=text/javascript"></script>
     <link rel="stylesheet" type="text/css" href="//2016.igem.org/Team:Slovenia/libraries/custom-css?action=raw&ctype=text/css">
+
     <link rel="stylesheet" type="text/css"
     <script type="text/javascript" src="//2016.igem.org/Team:Slovenia/libraries/custom-js?action=raw&ctype=text/javascript"></script>
+
          href="//2016.igem.org/Team:Slovenia/libraries/custom-css?action=raw&ctype=text/css">
 +
     <script type="text/javascript"
 +
            src="//2016.igem.org/Team:Slovenia/libraries/custom-js?action=raw&ctype=text/javascript"></script>
 
     <script type="text/javascript"
 
     <script type="text/javascript"
 
             src="//2016.igem.org/Team:Slovenia/libraries/zitator-js?action=raw&ctype=text/javascript"></script>
 
             src="//2016.igem.org/Team:Slovenia/libraries/zitator-js?action=raw&ctype=text/javascript"></script>
 
     <script type="text/javascript"
 
     <script type="text/javascript"
 
             src="https://2016.igem.org/Team:Slovenia/libraries/bibtexparse-js?action=raw&ctype=text/javascript"></script>
 
             src="https://2016.igem.org/Team:Slovenia/libraries/bibtexparse-js?action=raw&ctype=text/javascript"></script>
<!-- MathJax (LaTeX for the web) -->
+
    <!-- MathJax (LaTeX for the web) -->
 
     <script type="text/x-mathjax-config">
 
     <script type="text/x-mathjax-config">
 
         MathJax.Hub.Config({
 
         MathJax.Hub.Config({
Line 34: Line 36:
 
             SVG: { linebreaks: { automatic: true, width: "200% container" }}
 
             SVG: { linebreaks: { automatic: true, width: "200% container" }}
 
         });
 
         });
 +
   
 +
   
 
     </script>
 
     </script>
<script type="text/javascript" async
+
    <script type="text/javascript" async
 
             src="//2016.igem.org/common/MathJax-2.5-latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML">
 
             src="//2016.igem.org/common/MathJax-2.5-latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML">
 
     </script>
 
     </script>
 
</head>
 
</head>
 
<body>
 
<body>
 
 
<div id="example">
 
<div class="pusher">
 
<div class="full height">
 
<div class="banana">
 
<a href = "//2016.igem.org/Team:Slovenia">
 
<img class="ui medium sticky image" src="//2016.igem.org/wiki/images/d/d1/T--Slovenia--logo.png">
 
</a>
 
<div class="ui vertical sticky text menu">
 
<a class="item" href="#intro" style="margin-left: 10%">
 
<i class="selected radio icon"></i>
 
<b>Project</b>
 
</a>
 
<a class="item" href="#achievements" style="margin-left: 10%">
 
<i class="selected radio icon"></i>
 
<b>Achievements</b>
 
</a>
 
<a class="item" href="#requirements" style="margin-left: 10%">
 
<i class="selected radio icon"></i>
 
<b>Medal requirements</b>
 
</a>
 
<a class="item" href="idea">
 
<i class="chevron circle right icon"></i>
 
<b>Idea</b>
 
</a>
 
 
</div>
 
 
</div>
 
<div class="article" id="context">
 
<!-- menu goes here -->
 
<!-- content goes here -->
 
<div>
 
<div class="main ui citing justified container">
 
<h1 class = "ui centered dividing header"><span class="section">nbsp;</span>Reporters</h1>
 
<div class = "ui segment">
 
<p>As our project was aimed develop novel orthogonal signaling pathways based on proteases, as well as at the development of the protein ER retention and release system,
 
we tested and adapted several types of reporters, that will be useful for other iGEM teams.
 
</p>
 
<p>To measure the activity of the proteases we used three types of reporters based on the firefly luciferase: the cleavable fLuc, the circularly permutated fLuc (cpLuc)
 
and the cyclic fLuc (cycLuc). Additionally, we developed a split luciferase system that functions as an output of logic gates, which integrated the activity of orthogonal
 
proteases.
 
</p>
 
<p>Finally, to measure the ER protein retention and release, we used TagRFP and SEAP reporters.</p>
 
<p>Luciferase reporter of the proteolytic activity can be designed either to lead to the decrease of its activity due to proteolysis or to generate the activity by
 
cleavage. Cleavable luciferase assay is expected to be relatively insensitive as it can only detect if a large fraction of the luciferase has been degraded, typically
 
more than 20%, while an assay that leads to the activation of the luciferase might be able to detect much smaller fraction of the proteolytic cleavage.
 
</p>
 
<h4>Cleavable luciferase</h4><br/>
 
<p> Into the loop of the firefly luciferase (fLuc) we inserted amino acid sequence that is targeted by proteases. The substrate sequence thus divided the fLuc into
 
two fragments (nLuc and cLuc), with a protease cleavage site between them (<ref>1</ref>A and B). The insertion site for the substrate sequence was based on the
 
previously described split luciferase system <x-ref>Shekhawat2009</x-ref>, where we expected that this site would also be permissible to short linker insertion
 
without significantly altering luciferase activity. Upon addition of an appropriate protease, the reporter would be cleaved at the substrate site and the two
 
fragments would dissociate and in turn decrease the fLuc activity. In this system higher protease activity corresponds to a lower luciferase activity. The reporters
 
were additionally equipped with a protein tag at the N-terminal and C-terminal end in order to allow immunostaining to detect protein cleavage by the western blot
 
(<ref>1</ref>).
 
</p>
 
<div style = "float:left;">
 
<figure data-ref="1">
 
<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/4/44/T--Slovenia--4.9.4.png" >
 
<figcaption><b>INSERT!!</b><br/></figcaption>
 
</figure>
 
</div>
 
<h4 style="clear:both">Circularly permuted luciferase</h4><br/>
 
<p>The first reporter to measure protease activity that results in the generation of the luciferase activity by the proteolytic cleavage was a circularly permuted
 
version of the firefly luciferase (cpLuc). Luciferase is an oxidative enzyme that produces bioluminescence. The protein consists of two compact domains: the larger
 
N- and the smaller C-terminal domain. The C-terminal domain is connected to the N-terminal domain by a flexible hinge. When bound to the substrate luciferin, luciferase
 
has to undergo a conformational change from an open to a closed form with the two domains coming together to enclose the substrate and efficiently catalyze its oxidation
 
<x-ref>Conti</x-ref>.
 
</p>
 
<div style = "float:right;">
 
<figure data-ref="2">
 
<img onclick="resize(this);" class="ui medium image" src=" https://static.igem.org/mediawiki/2016/0/08/T--Slovenia--4.5.1.png" >
 
<figcaption><b>Proteolytic activity determined by the cpLuc reporters.</b><br/>
 
HEK293T cells were transfected with 100ng of indicated reporter constructs and 70ng of their corresponding proteases. Luciferase activity was measured 24h after transfection. The results are presented as normalized firefly luciferase activity (RLU).</figcaption>
 
</figure>
 
</div>
 
<p>The circularly permutated luciferase makes use of this requirement for a conformational change by rearranging the sequence of the protein. The permutation is obtained
 
by placing the C-terminal region of the protein (amino acids 234-544) upstream of the N-terminal region (amino acids 4-233) and connecting them by a short linker
 
(<ref>1</ref>C and D), which contains a protease cleavage site <x-ref>Fan2008</x-ref>. This linker prevents the conformational change required for the efficient
 
catalysis; therefore, the catalytic cycle is triggered only upon cleavage of the linker by a protease.
 
</p>
 
<p>Activity of the cpLuc depended on the protease cleavage for all four tested proteases as expected (<ref>2</ref>), however light emission from this reporter system was
 
relatively low, compelling us to look for another reporter.
 
</p>
 
 
<h4 style="clear:both">Cyclic luciferase</h4><br/>
 
<div style = "float:left;">
 
<figure data-ref="3">
 
<img onclick="resize(this);" class="ui medium image" src="  4.4.X" >
 
<figcaption><b> Luciferase reporters.</b><br/>
 
(A) Scheme of wild-type luciferase showing the site of insertion for the cleavage peptide (B) Scheme of cleavable luciferase and the mechanism of
 
inactivation by proteolysis (C) Scheme of wild-type luciferase showing the split site and the circular permutation (D) Scheme of circularly permuted
 
luciferase and the mechanism of activation by proteolysis. (E) Scheme of cyclic luciferase, cyclization by intein excision and mechanism of activation
 
by proteolysis.</figcaption>
 
</figure>
 
</div>
 
<p>The cyclic luciferase system takes the mechanism of cpLuc one step further by fusing two fragments of an intein to the ends of the cpLuc. Inteins are protein
 
fragments that allow protein splicing and cyclization by formation of a new peptide bond between the N- and C-termini of the protein, which generates an inactive
 
protein that can be activated by the proteolytic cleavage. We expected this reporter to result in a higher signal from the cpLuc due to the stabilization of the
 
protein by cyclization (<ref>3</ref>E). To further optimize the dynamic range of the system, a PEST sequence for fast digestion of the protein was included at
 
the C-terminus of the protein. This sequence targets any of the unspliced protein to degradation, while the spliced cyclic protein remains stable, since the PEST
 
sequence is excised along with the intein fragments during the splicing reaction <x-ref>Kanno2007</x-ref>.
 
</p>
 
 
<h4 style="clear:both">TagRFP</h4><br/>
 
<div style = "float:right;">
 
<figure data-ref="4">
 
<img onclick="resize(this);" class="ui medium image" src=" https://static.igem.org/mediawiki/2016/9/9e/T--Slovenia--6.2.1.png ">
 
<figcaption><b>INSERT!!</b><br/></figcaption>
 
</figure>
 
</div>
 
<p>For detection of the localization of our ER targeted reporters inside cells we used the fluorescent protein TagRFP (Evrogen), which was fused to the
 
appropriate localization and proteolytic target tags and used confocal microscopy for detection. Merzlyak et al. <x-ref> Merzlyak2007</x-ref>, modified the
 
wild type RFP from the sea anemone Entacmaea quadricolor to prolong its fluorescence lifetime and make it less susceptible to pH. With addition of the AU1 tag,
 
we were also able to detect its expression and secretion with western blot (<ref>4</ref>).
 
</p>
 
 
<h4 style="clear:both">SEAP</h4>
 
 
<p>For an assay of protein secretion with increased sensitivity we replaced the TagRFP with human secreted alkaline phosphatase (SEAP) reporter, which can
 
readily and accurately be quantified within the medium of transfected cells <x-ref>Berger1988</x-ref>. SEAP released into the medium was detected with
 
Quanti-blue colorimetric enzyme assay (Invivogen) (<ref>5</ref>).
 
<div style = "float:right;">
 
<figure data-ref="5">
 
<img onclick="resize(this);" class="ui medium image" src="https://static.igem.org/mediawiki/2016/6/61/T--Slovenia--6.2.2.png">
 
<figcaption><b>INSERT!!</b><br/></figcaption>
 
</figure>
 
</div>
 
</p><p style = "clear:right;"></p>
 
 
 
  
  
 +
<div id="example">
 +
    <div class="pusher">
 +
        <div class="full height">
 +
            <div class="banana">
 +
                <a href="//2016.igem.org/Team:Slovenia">
 +
                    <img class="ui medium sticky image" src="//2016.igem.org/wiki/images/d/d1/T--Slovenia--logo.png">
 +
                </a>
 +
                <div class="ui vertical sticky text menu">
 +
                    <a class="item" href="//2016.igem.org/Team:Slovenia/Protease_signaling/Overview">
 +
                        <i class="chevron circle left icon"></i>
 +
                        <b>Overview</b>
 +
                    </a>
 +
                    <a class="item" href="//2016.igem.org/Team:Slovenia/Protease_signaling/Reporters" style="color:#DB2828;">
 +
                        <i class="selected radio icon"></i>
 +
                        <b>Reporters</b>
 +
                    </a>
 +
                    <a class="item" href="#ach" style="margin-left: 10%">
 +
                        <i class="selected radio icon"></i>
 +
                        <b>Achievements</b>
 +
                    </a>
 +
                    <a class="item" href="#mot" style="margin-left: 10%">
 +
                        <i class="selected radio icon"></i>
 +
                        <b>Introduction</b>
 +
                    </a>
 +
                    <a class="item" href="#cle" style="margin-left: 10%">
 +
                        <i class="selected radio icon"></i>
 +
                        <b>Cleavable luciferase</b>
 +
                    </a>
 +
                    <a class="item" href="#cir" style="margin-left: 10%">
 +
                        <i class="selected radio icon"></i>
 +
                        <b>Circularly permuted</b>
 +
                        <br/>
 +
                        <b style="margin-left: 12%">luciferase</b>
 +
                    </a>
 +
                    <a class="item" href="#cyc" style="margin-left: 10%">
 +
                        <i class="selected radio icon"></i>
 +
                        <b>Cyclic luciferase</b>
 +
                    </a>
 +
                    <a class="item" href="#tag" style="margin-left: 10%">
 +
                        <i class="selected radio icon"></i>
 +
                        <b>Tag RFP</b>
 +
                    </a>
 +
                    <a class="item" href="#seap" style="margin-left: 10%">
 +
                        <i class="selected radio icon"></i>
 +
                        <b>SEAP</b>
 +
                    </a>
 +
                    <a class="item" href="//2016.igem.org/Team:Slovenia/Protease_signaling/Orthogonality">
 +
                        <i class="chevron circle right icon"></i>
 +
                        <b>Orthogonality</b>
 +
                    </a>
 +
 +
                </div>
 +
 +
            </div>
 +
            <div class="article" id="context">
 +
                <!-- menu goes here -->
 +
                <!-- content goes here -->
 +
                <div>
 +
                    <div class="main ui citing justified container">
 +
                        <div>
 +
                            <h1 class="ui left dividing header"><span id="ach" class="section colorize">nbsp;</span>Protease
 +
                                reporters</h1>
 +
                            <div class="ui segment" style="background-color: #ebc7c7; ">
 +
                                <p><b>
 +
                                    <ul>
 +
                                        <li>Three luciferase based reporters for detection of protease activity were
 +
                                            designed and tested.
 +
                                        <li>Cleavable firefly luciferase was used as an inverse reporter, displaying a
 +
                                            decrease in luminescence upon proteolytic cleavage.
 +
                                        <li>Circularly permuted and cyclic luciferase were used as direct reporters,
 +
                                            displaying an increase in luminescence upon proteolytic cleavage.
 +
                                        <li>Fluorescent and enzymatic reporters with cleavable ER retention signals were
 +
                                            designed to test induced protein secretion.
 +
                                    </ul>
 +
                                </b></p>
 +
                            </div>
 +
                        </div>
 +
                        <div class="ui segment">
 +
                            <h4><span id="mot" class="section colorize">nbsp;</span></h4>
 +
                            <p>As our project was aimed to develop novel orthogonal signaling pathways based on
 +
                                proteases, as well as at the development of a protein ER retention and release system,
 +
                                we tested and adapted several types of reporters, that will also be useful to other iGEM
 +
                                teams.
 +
                            </p>
 +
                            <p>To measure the activity of the proteases, we used three types of reporters based on
 +
                                firefly luciferase: the cleavable fLuc inverse reporter, the circularly permutated fLuc
 +
                                (cpLuc)
 +
                                and the cyclic fLuc (cycLuc).</p>
 +
 +
                            <p>Luciferase reporters for detection of proteolytic activity can be designed to lead either
 +
                                to a decrease of its activity due to proteolysis (inverse reporter) or to increase
 +
                                luciferase activity by proteolytic
 +
                                cleavage of an inactive enzyme. The cleavable luciferase inverse reporter assay is
 +
                                expected to be relatively insensitive as a large fraction of the luciferase, typically
 +
                                more than 20%, has to be degraded before an appreciable difference in the output is
 +
                                detected, while an assay that leads to activation of luciferase might be able to detect
 +
                                much proteolytic activity.
 +
                            </p>
 +
                            <div style="float:left; width:100%">
 +
                                <figure data-ref="3">
 +
                                    <img src="https://static.igem.org/mediawiki/2016/a/a6/T--Slovenia--4.4.x.png">
 +
                                    <figcaption><b> Luciferase reporters.</b><br/>
 +
                                        <p style="text-align:justify">(A) Scheme of wild-type luciferase. The site of
 +
                                            insertion for the cleavage peptide is marked by an arrow. (B) Scheme of
 +
                                            cleavable luciferase and the mechanism of
 +
                                            inactivation by proteolysis. (C) Scheme of wild-type luciferase. The split
 +
                                            site and the circular permutation are marked by arrows. (D) Scheme of
 +
                                            circularly permuted
 +
                                            luciferase and the mechanism of activation by proteolysis. (E) Scheme of
 +
                                            cyclic luciferase, cyclization by intein excision and mechanism of
 +
                                            activation
 +
                                            by proteolysis.
 +
                                        </p>
 +
                                    </figcaption>
 +
                                </figure>
 +
                            </div>
 +
                            <p>
 +
                                Additionally, we developed a split luciferase system that functions as an output for
 +
                                logic functions, integrating the activity of orthogonal
 +
                                proteases. Finally, to measure protein retention and release from the endoplasmic
 +
                                reticulum (ER), we used TagRFP and SEAP reporters.
 +
                            </p>
 +
                        </div>
 +
 +
                        <h1><span class="section colorize">nbsp;</span>Results</h1>
 +
                        <div class="ui segment">
 +
                            <div>
 +
                                <h3><span id="cle" class="section colorize">nbsp;</span>Cleavable luciferase</h3>
 +
                                <p> We inserted amino acid sequences targeted by selected proteases into a loop of the
 +
                                    firefly luciferase. The substrate sequence thus divided the fLuc into
 +
                                    two fragments (nLuc and cLuc), with a protease cleavage site between them (
 +
                                    <ref>3</ref>A and B). The insertion site for the substrate sequence was based on the
 +
                                    previously described split luciferase system
 +
                                    <x-ref>Shekhawat2009</x-ref>
 +
                                    , where we expected that this site would also be permissible to short linker
 +
                                    insertion
 +
                                    without significantly altering luciferase activity. Upon addition of an appropriate
 +
                                    protease, the reporter would be cleaved at the substrate site and the two
 +
                                    fragments would dissociate, in turn decreasing the fLuc activity. In this system
 +
                                    higher protease activity corresponds to a lower luciferase activity. The reporters
 +
                                    were additionally equipped with protein tags at the N- and C-termini in order to
 +
                                    allow immunostaining for detection of protein cleavage by western blotting (<a
 +
                                            href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Light_dependent_mediator#cleavable luciferase">Light-dependent
 +
                                        mediator Figure 5D</a>).
 +
                                </p>
 +
                            </div>
 +
                            <div>
 +
                                <h3 style="clear:both"><span id="cir" class="section colorize">nbsp;</span>Circularly permuted
 +
                                    luciferase</h3>
 +
                                <p>The first reporter to measure protease activity that results in generation of
 +
                                    luciferase activity by proteolytic cleavage was a circularly permuted
 +
                                    version of the firefly luciferase (cpLuc). Luciferase is an oxidative enzyme that
 +
                                    produces bioluminescence. The protein consists of two compact domains: the larger
 +
                                    N- and the smaller C-terminal domain. The C-terminal domain is connected to the
 +
                                    N-terminal domain by a flexible hinge. When bound to the substrate luciferin,
 +
                                    luciferase
 +
                                    has to undergo a conformational change from an open to a closed form with the two
 +
                                    domains coming together to enclose the substrate and efficiently catalyze its
 +
                                    oxidation
 +
                                    <x-ref>Conti</x-ref>
 +
                                    .
 +
                                </p>
 +
                                <div style="float:right; width:70%">
 +
                                    <figure data-ref="2">
 +
                                        <img src=" https://static.igem.org/mediawiki/2016/0/08/T--Slovenia--4.5.1.png">
 +
                                        <figcaption><b>Proteolytic activity determined by the cpLuc reporters.</b><br/>
 +
                                            <p style="text-align:justify">HEK293T cells were transfected with 100 ng of
 +
                                                indicated reporter constructs and 70 ng of their corresponding
 +
                                                proteases.
 +
                                                Luciferase activity was measured 24 h after transfection. The results
 +
                                                are presented as normalized firefly luciferase activity (RLU).
 +
                                            </p>
 +
                                        </figcaption>
 +
                                    </figure>
 +
                                </div>
 +
                                <p>The circularly permutated luciferase makes use of this requirement for a
 +
                                    conformational change by rearranging the sequence of the protein. The permutation is
 +
                                    obtained
 +
                                    by placing the C-terminal region of the protein (amino acids 234-544) upstream of
 +
                                    the N-terminal region (amino acids 4-233) and connecting them by a short linker
 +
                                    (<ref>3</ref>C and D), which contains a protease cleavage site
 +
                                    <x-ref>Fan2008</x-ref>
 +
                                    . This linker prevents the conformational change required for efficient
 +
                                    catalysis; therefore, the catalytic cycle is triggered only upon cleavage of the
 +
                                    linker by a protease.
 +
                                </p>
 +
                                <p>We designed and tested four new cpLuc reporters. Activity of all four reporters
 +
                                    depended on cleavage by the corresponding protease as expected (<ref>2</ref>), however light emission from this reporter system was
 +
                                    relatively low, compelling us to look for a more efficient reporter.
 +
                                </p>
 +
                            </div>
 +
                            <div>
 +
                                <h3 style="clear:both"><span id="cyc" class="section colorize">nbsp;</span>Cyclic luciferase</h3>
 +
                                <p>The cyclic luciferase system takes the mechanism of cpLuc one step further by fusing
 +
                                    two fragments of an intein to the ends of the cpLuc. Inteins are protein
 +
                                    fragments that allow protein splicing and cyclization by formation of a new peptide
 +
                                    bond between the N- and C-termini of the protein, which generates an inactive
 +
                                    protein that can be activated by the proteolytic cleavage. We expected this reporter
 +
                                    to result in a higher signal from the cpLuc due to the stabilization of the
 +
                                    protein by cyclization (
 +
                                    <ref>3</ref>E). To further optimize the dynamic range of the system, a PEST sequence for fast
 +
                                    digestion of the protein was included at
 +
                                    the C-terminus of the protein. This sequence targets any of the unspliced protein to
 +
                                    degradation, while the spliced cyclic protein remains stable, since the PEST
 +
                                    sequence is excised along with the intein fragments during the splicing reaction
 +
                                    <x-ref>Kanno2007</x-ref>
 +
                                    .
 +
                                </p>
 +
                                <p style="clear:both;"></p>
 +
                            </div>
 +
                            <div>
 +
                                <h3 style="clear:both"><span id="tag" class="section colorize">nbsp;</span>TagRFP</h3>
 +
                                <p>For detection of the localization of our ER targeted reporters inside cells we used
 +
                                    the fluorescent protein TagRFP (Evrogen), which was fused to the
 +
                                    appropriate localization and proteolytic target tags and used confocal microscopy
 +
                                    for detection. Merzlyak et al.
 +
                                    <x-ref>Merzlyak2007</x-ref>
 +
                                    , modified the
 +
                                    wild type RFP from the sea anemone <i>Entacmaea quadricolor</i> to prolong its
 +
                                    fluorescence lifetime and make it less susceptible to pH. With addition of the AU1
 +
                                    tag,
 +
                                    we were also able to detect its expression and secretion with western blot (<a
 +
                                            href="https://2016.igem.org/Team:Slovenia/Demonstrate#tagRFP">Protease
 +
                                        inducible secretion Figure 3</a>).
 +
                                </p>
 +
                            </div>
 +
                            <div>
 +
                                <h3 style="clear:both"><span id="seap" class="section colorize">nbsp;</span>SEAP</h3>
  
 +
                                <p>For a protein secretion assay with increased sensitivity we replaced the TagRFP with
 +
                                    the human secreted alkaline phosphatase (SEAP) reporter, which can
 +
                                    readily and accurately be quantified within the medium of transfected cells
 +
                                    <x-ref>Berger1988</x-ref>
 +
                                    . SEAP released into the medium was detected with the
 +
                                    Quanti-blue colorimetric enzyme assay (Invivogen) (<a
 +
                                            href="https://2016.igem.org/Team:Slovenia/Demonstrate#SEAP">Protease
 +
                                        inducible secretion Figure 4</a>).
  
</div>
+
                                </p>
</div>
+
                                <p style="clear:left;"></p>
+
                            </div>
+
                        </div>
</div>
+
                        <h3 class="ui left dividing header"><span id="ref-title" class="section colorize">&nbsp;</span>References
</div>
+
                        </h3>
</div>
+
                        <div class="ui segment citing" id="references"></div>
</div>
+
                    </div>
</div>
+
                </div>
 +
            </div>
 +
        </div>
 +
    </div>
 +
</div>
 +
<div>
 +
    <a href="//igem.org/Main_Page">
 +
        <img border="0" alt="iGEM" src="//2016.igem.org/wiki/images/8/84/T--Slovenia--logo_250x250.png" width="5%"
 +
            style="position: fixed; bottom:0%; right:1%;">
 +
    </a>
 +
</div>
 
</body>
 
</body>
 
</html>
 
</html>

Latest revision as of 14:57, 19 October 2016

Reporters

nbsp;Protease reporters

  • Three luciferase based reporters for detection of protease activity were designed and tested.
  • Cleavable firefly luciferase was used as an inverse reporter, displaying a decrease in luminescence upon proteolytic cleavage.
  • Circularly permuted and cyclic luciferase were used as direct reporters, displaying an increase in luminescence upon proteolytic cleavage.
  • Fluorescent and enzymatic reporters with cleavable ER retention signals were designed to test induced protein secretion.

nbsp;

As our project was aimed to develop novel orthogonal signaling pathways based on proteases, as well as at the development of a protein ER retention and release system, we tested and adapted several types of reporters, that will also be useful to other iGEM teams.

To measure the activity of the proteases, we used three types of reporters based on firefly luciferase: the cleavable fLuc inverse reporter, the circularly permutated fLuc (cpLuc) and the cyclic fLuc (cycLuc).

Luciferase reporters for detection of proteolytic activity can be designed to lead either to a decrease of its activity due to proteolysis (inverse reporter) or to increase luciferase activity by proteolytic cleavage of an inactive enzyme. The cleavable luciferase inverse reporter assay is expected to be relatively insensitive as a large fraction of the luciferase, typically more than 20%, has to be degraded before an appreciable difference in the output is detected, while an assay that leads to activation of luciferase might be able to detect much proteolytic activity.

Luciferase reporters.

(A) Scheme of wild-type luciferase. The site of insertion for the cleavage peptide is marked by an arrow. (B) Scheme of cleavable luciferase and the mechanism of inactivation by proteolysis. (C) Scheme of wild-type luciferase. The split site and the circular permutation are marked by arrows. (D) Scheme of circularly permuted luciferase and the mechanism of activation by proteolysis. (E) Scheme of cyclic luciferase, cyclization by intein excision and mechanism of activation by proteolysis.

Additionally, we developed a split luciferase system that functions as an output for logic functions, integrating the activity of orthogonal proteases. Finally, to measure protein retention and release from the endoplasmic reticulum (ER), we used TagRFP and SEAP reporters.

nbsp;Results

nbsp;Cleavable luciferase

We inserted amino acid sequences targeted by selected proteases into a loop of the firefly luciferase. The substrate sequence thus divided the fLuc into two fragments (nLuc and cLuc), with a protease cleavage site between them ( 3A and B). The insertion site for the substrate sequence was based on the previously described split luciferase system Shekhawat2009 , where we expected that this site would also be permissible to short linker insertion without significantly altering luciferase activity. Upon addition of an appropriate protease, the reporter would be cleaved at the substrate site and the two fragments would dissociate, in turn decreasing the fLuc activity. In this system higher protease activity corresponds to a lower luciferase activity. The reporters were additionally equipped with protein tags at the N- and C-termini in order to allow immunostaining for detection of protein cleavage by western blotting (Light-dependent mediator Figure 5D).

nbsp;Circularly permuted luciferase

The first reporter to measure protease activity that results in generation of luciferase activity by proteolytic cleavage was a circularly permuted version of the firefly luciferase (cpLuc). Luciferase is an oxidative enzyme that produces bioluminescence. The protein consists of two compact domains: the larger N- and the smaller C-terminal domain. The C-terminal domain is connected to the N-terminal domain by a flexible hinge. When bound to the substrate luciferin, luciferase has to undergo a conformational change from an open to a closed form with the two domains coming together to enclose the substrate and efficiently catalyze its oxidation Conti .

Proteolytic activity determined by the cpLuc reporters.

HEK293T cells were transfected with 100 ng of indicated reporter constructs and 70 ng of their corresponding proteases. Luciferase activity was measured 24 h after transfection. The results are presented as normalized firefly luciferase activity (RLU).

The circularly permutated luciferase makes use of this requirement for a conformational change by rearranging the sequence of the protein. The permutation is obtained by placing the C-terminal region of the protein (amino acids 234-544) upstream of the N-terminal region (amino acids 4-233) and connecting them by a short linker (3C and D), which contains a protease cleavage site Fan2008 . This linker prevents the conformational change required for efficient catalysis; therefore, the catalytic cycle is triggered only upon cleavage of the linker by a protease.

We designed and tested four new cpLuc reporters. Activity of all four reporters depended on cleavage by the corresponding protease as expected (2), however light emission from this reporter system was relatively low, compelling us to look for a more efficient reporter.

nbsp;Cyclic luciferase

The cyclic luciferase system takes the mechanism of cpLuc one step further by fusing two fragments of an intein to the ends of the cpLuc. Inteins are protein fragments that allow protein splicing and cyclization by formation of a new peptide bond between the N- and C-termini of the protein, which generates an inactive protein that can be activated by the proteolytic cleavage. We expected this reporter to result in a higher signal from the cpLuc due to the stabilization of the protein by cyclization ( 3E). To further optimize the dynamic range of the system, a PEST sequence for fast digestion of the protein was included at the C-terminus of the protein. This sequence targets any of the unspliced protein to degradation, while the spliced cyclic protein remains stable, since the PEST sequence is excised along with the intein fragments during the splicing reaction Kanno2007 .

nbsp;TagRFP

For detection of the localization of our ER targeted reporters inside cells we used the fluorescent protein TagRFP (Evrogen), which was fused to the appropriate localization and proteolytic target tags and used confocal microscopy for detection. Merzlyak et al. Merzlyak2007 , modified the wild type RFP from the sea anemone Entacmaea quadricolor to prolong its fluorescence lifetime and make it less susceptible to pH. With addition of the AU1 tag, we were also able to detect its expression and secretion with western blot (Protease inducible secretion Figure 3).

nbsp;SEAP

For a protein secretion assay with increased sensitivity we replaced the TagRFP with the human secreted alkaline phosphatase (SEAP) reporter, which can readily and accurately be quantified within the medium of transfected cells Berger1988 . SEAP released into the medium was detected with the Quanti-blue colorimetric enzyme assay (Invivogen) (Protease inducible secretion Figure 4).

 References