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<h2>Abstract</h2>
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<p>The UNSW iGEM team designed a new IaaH encoding part for improved functionality of the indole-3-aectamide hydrolase (IaaH) enzyme. Improving on the <a target="_blank" href="http://parts.igem.org/Part:BBa_K1789001">BBa_K89001</a> part submitted by the 2015 NUDT_China team, a HisTag and GSG linker were added to the N-terminus of the enzyme, and a SpyTag and GSG linker was added to the C-terminus. Following these additions, the team was able to successfully express and purify the new part utilising the HisTag's affinity for nickel ions to purify the enzyme using IMAC purification. We also demonstrated the ability of IaaH with SpyTag to covalently bind with SpyCatcher proteins through SDS-PAGE. More details can be found on the improved part's registry page at <a target="_blank" href="http://parts.igem.org/Part:BBa_K2710005">BBa_K2710005</a>. Through these improvements, we have expanded the utility of this part twofold. First, the protein can be easily purified from cell lysates, and secondly, it now includes an irreversible attachment mechanism, robust over a range of conditions. </p>
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<h2>Overview</h2>
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<p>Protein scaffold components and proteins that attach to the scaffold must be expressed and purified for self-assembly and enzyme activity experiments. Sequence-verified plasmids were heat shock transformed into <em>Escherichia coli</em> cells and expressed for recombinant protein production. The proteins were then purified from cell lysates with Immobilised Metal Affinity Chromatography (IMAC). Nine proteins have been successfully expressed and purified, enabling the construction of our scaffold-enzyme complex.</p>
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<h2>Introduction</h2>
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<h2>The Enzyme</h2>
 +
<p> <b>Indole-3-acetamide hydrolase (IaaH)</b> is an enzyme involved in the biosynthesis of indole-3-aectic acid. IaaH originating from <i>Alcaligenes sp.</i> strain HPC127114 was fused with a SpyTag and a hexahistidine tag (HisTag)<sup>1</sup>. </p>
  
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<p>A large range of proteins are required for the construction and characterisation of our scaffold-enzyme complex. The scaffold consists of the molecular chaperones alpha prefoldin (aPFD) and beta prefoldin (bPFD), derived from <em>Methanobacterium thermoautotrophicum</em><sup><a href="#references">1</a></sup>, fused with SpyCatcher<sup><a href="#references">2</a></sup> (SpyC) and SnoopCatcher<sup><a href="#references">3</a></sup> (SnoopC) respectively on their C-termini. These self-assemble to form a hexameric complex that is able to covalently bind SpyTags and SnoopTags. Two enzymes were designed for attachment to the scaffold: the enzymes tryptophan 2-monooxygenase (IaaM) originating from <em>Pseudomonas savastanoi</em><sup><a href="#references">4</a></sup> fused with a SnoopTag (SnoopT) and indole acetamide hydrolase (IaaH) originating from <em>Alcaligenes sp.</em> Strain HPC1271<sup><a href="#references">5</a></sup> fused with a SpyTag (SpyT). In addition, the fluorescent proteins mVenus and mCerulean3<sup><a href="#references">6</a></sup> were fused with a SpyTag and SnoopTag respectively. All proteins have been expressed with a 6xHis-Tag, utilising the affinity of the HisTag for nickel ions for IMAC purification<sup><a href="#references">7</a></sup>.</p>
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<p>The auxin indole-3-acetic acid, is a plant hormone involved in the regulation of plant growth and development<sup>2</sup>. Indole-3-aecetic acid can be synthesised via the indole-3-acetamide pathway, which converts tryptophan to indole-3-aectic acid in a two-step enzymatic pathway<sup>3</sup> <b>(Figure 1)</b>. The flavoprotein tryptophan 2-monooxygenase (IaaM) catalyses the oxidative decarboxylation of tryptophan to indole-3-acetamide in the first, rate limiting step of the pathway<sup>4</sup>. Subsequently, the enzyme indole-3-acetamide hydrolase (IaaH) converts indole-3-acetamide to indole-3-aectic acid<sup>1</sup>.</p>
  
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<p>Initial attempts to express these proteins in <em>E. coli</em> using the pET-Duet-1 and pRSF-Duet-1 vectors were unsuccessful, likely due to our design of the plasmids. Following the subcloning of the inserts into a new vector, pET-19b, 5 of these proteins were successfully expressed and purified. In addition, strains of <em>Escherichia coli</em> (<em>E. coli</em>) containing plasmids for His-tagged mVenus, mCerulean3, gamma prefoldin with a C-terminal SpyC (gPFD-SpyC) and gamma prefoldin with an N- and a C-terminal SpyC (SpyC-gPFD-SpyC) were used for protein expression and purification.</p>
 
  
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<p>Purification of these proteins enables characterisation of the self-assembly of the scaffold-enzyme complex with Size Exclusion Chromatography (SEC), SDS-PAGE and Transmission Electron Microscopy (TEM) and characterisation of assembly conditions, measurement of enzyme activity with the Salkowski assay and HPLC and characterisation of the distance between attachment sites with F&ouml;rster resonance energy transfer (FRET).</p>
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<br />
  
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<p>We have developed a robust method for recombinant expression and purification of our novel protein scaffold. The purity of the proteins was investigated with SDS-PAGE, which is essential to ensure the quality and accuracy of all experimental characterisation of our scaffold-enzyme complex.</p>
 
  
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<h2>Aims</h2>
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<img src=https://static.igem.org/mediawiki/2018/7/78/T--UNSW_Australia--bec-design-iaa-pathway.png>
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<p class=figure-legend><b>Figure 1:</b> The indole-3-acetamide pathway for indole-3-aecetic acid biosynthesis.</p>
  
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<p>To produce, purify and characterise prefoldin scaffold proteins and proteins that attach to our scaffold for further experiments on the stability, assembly and efficacy of our enzyme scaffold.</p>
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<br />
 +
<h2>Improvements</h2>
 +
<p> The original part was improved by two key additions; a <b>SpyTag</b> and a <b>HisTag</b>.
  
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<h2>Methods</h2>
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<p>The <b>SpyTag</b> forms one component of the SpyTag/SpyCatcher system, which enables covalent attachment of two proteins<sup>6</sup>. The SpyTag and SpyCatcher system was created by cleaving the CnaB2 domain of the fibronectin-binding protein FbaB derived from <i>Streptococcus pyogenes</i> to form a thirteen residue SpyTag peptide and a 116-residue SpyCatcher peptide<sup>6</sup>. The SpyTag (1.1 kDa) and SpyCatcher (12 kDa) form an irreversible intramolecular isopeptide bond between Asp<sup>117</sup> on SpyTag and Lys<sup>31</sup> on SpyCatcher<sup>6</sup>, spontaneously and specifically binding to each other so that they can be used as attachment mechanisms to create new, self-assembling protein arrangements<sup>6</sup> <b>(Figure 2)</b>.</p>
 +
<p>It is particularly useful because neither component needs to be at the C or N terminus<sup>7</sup>, and the effect on the attached protein’s activity appears to be negligible<sup>8</sup>. It also reported as useful in a variety of reaction conditions, with Howarth showing that the SpyTag/SpyCatcher “had a high yield...required only simple mixing (and) tolerated diverse conditions (pH, buffer components and temperature)”<sup>9</sup>. </p>
  
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<p><em>Escherichia coli</em> T7 Express cells (NEB) were heat shock transformed with a plasmid containing the gene of interest.  The bacteria were grown in Luria broth (LB) media with ampicillin at 37<sup>o</sup>C at 200 rpm, induced with isopropyl &beta;-D-1-thiogalactopyranoside (IPTG) at 1 mM when the OD<sub>600</sub> of the media reached 0.6 and then grown overnight at room temperature. The cell pellet was collected by centrifugation and lysed by sonication. The cell lysate was then centrifuged to remove cell debris, and only the soluble fraction was collected. The soluble fraction was loaded onto a HisTrap HP 1 mL column (GE Healthcare) and purified using immobilised metal affinity chromatography (IMAC). Elutions were analysed with SDS-PAGE and buffer exchanged into PBS pH 8 using Pierce Protein Concentrators PES, 10K MWCO, 2-6 mL (Thermo Scientific) or by dialysis. The concentration of buffer exchanged proteins were then quantified by the bicinchoninic acid (BCA) assay.</p>
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<p class="figure-legend"><b>Figure 1:</b> Summary of methods for protein expression and purification.</p>
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<p class=figure-legend><b>Figure 2:</b> A spontaneous isopeptide bond forms between SpyTag and SpyCatcher. Image created using PDB ID: 4MLS<sup>10</sup> </p>
  
 +
<br />
 +
<p>A <b>HisTag</b> (six consecutive histidine residues, also known as a hexahistidine tag) was added to IaaH to enable purification, utilising the affinity of the HisTag for nickel ions for Immobilised Metal Affinity Chromatography purification<sup>5</sup>.</p>
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<p>Detailed protocols can be found <a target="_blank" href="https://2018.igem.org/Team:UNSW_Australia/Experiments">on our experiments page</a>.</p>
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<h2>Experimental Characterisation</h2>
 +
<p> The improved part was validated by sequence verification and a diagnostic gel.  Following this, the original and improved part were cloned into the expression vector pET19B and successfully expressed in <i>E. coli</i> T7 Express cells (NEB). The HisTag and SpyTag improvements were experimentally characterised through SDS-PAGE analysis of IMAC purifications and attachment to SpyCatcher fusion proteins. Refer to the <a target="_blank" href="http://parts.igem.org/Part:BBa_K2710005">BBa_K2710005</a> registry page for details of the experimental characterisation.</p>
  
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<h2>Results</h2>
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<p>Through these improvements, we have expanded the utility of this part twofold. First, the protein can be easily purified from cell lysates, and secondly, it now includes an irreversible attachment mechanism, robust over a range of conditions. </p>
  
−
<p>Following the successful subcloning of inserts from pET-Duet1, pRSF-Duet1 or pSB1C3 into pET19b, 9 proteins were successfully purified and analysed by SDS-PAGE. In addition, 3 parts from the Registry of Standard Biological Parts (BBa_K1789000, BBa_K1789001 and BBa_K515100) were expressed, but were not purified. These parts contained the enzymes IaaM and IaaH without His-tags.</p>
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<p>Furthermore, as wild-type gPFD polymerises to form filamentous structures, Transmission Electron Microscopy (TEM) was performed on gPFD-SpyC attached to IaaH-SpyT to investigate the effect of the attachment of the enzyme on filament formation. Attachment via the SpyTag/SpyCatcher mechanism appears to distort the filaments <b>(Figure 3)</b>.</p>
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<p class="figure-legend"><b>Figure 2:</b> SDS-PAGE analysis of IMAC purifications of His-tagged proteins. <b>A:</b> mVenus (MW: 27 kDa). <b>B:</b> mCerulean3 (MW: 27 kDa). <b>C:</b> gPFD-SpyC (MW: 31 kDa). <b>D:</b> SpyC-gPFD-SpyC (MW: 46 kDa). <b>E:</b> aPFD (MW: 17 kDa) (left) and bPFD (MW: 15 kDa) (right). <b>F:</b> bPFD-SnoopC (MW: 28 kDa). <b>G:</b> IaaH (without His-tag, unsuccessful purification) (MW: 49 kDa) (left) and IaaH-SpyT (MW: 53 kDa) (right). <b>H:</b> aPFD-SpyC (MW: 30 kDa). SeeBlue Plus 2 Pre-stained Protein Standard (Invitrogen) was used as the molecular weight standard for all SDS-PAGE analysis. Lanes are labelled as cell lysate (L), flow through (FT), wash (W) and elutions (E1, E2, E3, E4, E5).</p>
+
<p class="figure-legend fig5-leg"><b>Figure 3: </b>TEM demonstrates that gPFD and gPFD mutants are able to assemble and form filaments. A: Wild-type gPFD filaments. B: gPFD-SpyC filaments, indicating that gPFD is able to form filaments when fused to a SpyCatcher. C: gPFD-SpyC reacted with IaaH-SpyT. Clumps of filaments, or curled filaments were observed.</p>
  
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<h2>Discussion</h2>
 
  
−
<p>Initial attempts at protein expression were unsuccessful using pET-Duet-1 and pRSF-Duet-1 plasmids. After cloning our desired inserts into these plasmids, we attempted to express these proteins, but no expression could be detected by SDS-PAGE or Western Blot. Both <em>E. coli</em> T7 Express and Lemo21(DE3) cell lines were used for expression, and tested with 0.1 mM, 0.4 mM and 1 mM IPTG inductions. We hypothesised that the design of our plasmids inhibited expression, as the BioBrick prefix was placed between the ribosome binding site and the start codon of our coding sequence. We decided to subclone our inserts into pET-19b and remove the BioBrick prefix and suffix before retrying protein expression and purification.</p>
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<div id=references>
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<p>Despite these difficulties, 9 proteins were successfully purified, and 3 protein constructs from the iGEM registry were expressed. The following constructs were successfully purified:</p>
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<ul>
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<li>His-aPFD & His-bPFD – for assembly of the aPFD/bPFD hexamer, and as a negative control for the effect of scaffolding on enzyme activity.</li>
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<li>His-aPFD-SpyCatcher & His-bPFD-SnoopCatcher – the scaffold components of our complex that can covalently attach Spy-Tagged and Snoop-Tagged enzymes</li>
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<li>His-mVenus & His-mCerulean3 – for FRET experiments to investigate the distance between proteins attached to the scaffold.</li>
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<li>His-gPFD-SpyCatcher & His-SpyCatcher-gPFD-SpyCatcher – filamentous variants of prefoldin fused with SpyCatchers, to test SpyTag/SpyCatcher reactions and to determine if gPFD can form filaments with enzymes attached to its N- and/or C- terminus.</li>
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<li>His-IaaH-SpyTag – the second enzyme of our reaction pathway, indole-3-acetamide hydrolase, fused with a SpyTag for attachment to His-aPFD-SpyCatcher.</li>
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</ul>
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<p>The following BioBricks were expressed for comparison with the tagged versions of the enzymes:</p>
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<ul>
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<li>BBa_K1789000 – IaaM</li>
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<li>BBa_K1789001 – IaaH</li>
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<li>BBa_K515100 – IaaM and IaaH under a Pveg2 promoter</li>
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</ul>
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<p>Following protein purification, we encountered issues with solubility for some proteins. This was likely due to non-optimal buffer conditions or high concentrations of protein. Further experimentation and optimisation is required to identify the range of conditions in which these proteins are stable and able to assemble. In the future, we would like to express and purify all of our successfully cloned constructs, including the first enzyme of our reaction pathway, tryptophan-2-monooxygenase (IaaM), fused to the SnoopTag, fluorescent proteins with appropriate tags for FRET experiments and enzymes from other pathways fused with SpyTag and SnoopTag. In addition, we would like to increase the purity of our purifications and attempt larger scale protein expressions. These provide materials that are fundamental for the characterisation of the assembly of our scaffold, the distance between the attachment site, the rate of indole acetic acid production and the modularity of our system.</p>
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<h2>References</h2>
 
<h2>References</h2>
 
<ol>
 
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<li>Siegert, R., Leroux, M. R., Scheufler, C., Hartl, F. U. & Moarefi, I. Structure of the molecular chaperone prefoldin: unique interaction of multiple coiled coil tentacles with unfolded proteins. <em>Cell</em> <b>103</b>, 621–32 (2000).</li>
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<li>Mishra, P., Kaur, S., Sharma, A. N. and Jolly, R. S. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid. <i>PLoS One 11</i>, e0159009 (2016).</li>
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<li>Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. <em>Proc. Natl. Acad. Sci.</em> <b>109</b>, E690–E697 (2012).</li>
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<li>Davies, P. J. Plant Hormones: Biosynthesis, Signal Transduction, Action! ,  (Springer Netherlands, 2007).</li>
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<li>Veggiani, G. et al. Programmable polyproteams built using twin peptide superglues. <em>Proc. Natl. Acad. Sci.</em> <b>113</b>, 1202–1207 (2016).</li>
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<li>Spaepen, S., Vanderleyden, J. and Remans, R. Indole-3-acetic acid in microbial and microorganism-plant signaling. <i>FEMS Microbiol Rev</i>, <b>31</b> 425-448, doi:10.1111/j.1574-6976.2007.00072.x (2007).</li>
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<li>Gaweska, H. M., Taylor, A. B., Hart, P. J. & Fitzpatrick, P. F. Structure of the flavoprotein tryptophan 2-monooxygenase, a key enzyme in the formation of galls in plants. <em>Biochemistry</em> <b>52</b>, 2620–6 (2013).</li>
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<li>Gaweska, H. M., Taylor, A. B., Hart, P. J. and Fitzpatrick, P. F. Structure of the flavoprotein tryptophan 2-monooxygenase, a key enzyme in the formation of galls in plants. <em>Biochemistry</em>. <b>52</b> 2620–6 (2013).</li>
−
<li>Mishra, P., Kaur, S., Sharma, A. N. & Jolly, R. S. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid. <em>PLoS One</em> <b>11</b>, e0159009 (2016).</li>
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<li>Hochuli, E., Dobeli, H. and Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. <i>J Chromatogr.</i> <b>411</b> 177-184 (1987).</li>
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<li>Markwardt, M. L. et al. An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching. <em>PLoS One</em> <b>6</b>, e17896 (2011).</li>
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<li>Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. <i>Proc Natl Acad Sci U S A.</i> <b>109</b> E690-697, doi:10.1073/pnas.1115485109 (2012).</li>
−
<li>Hochuli, E., Bannwarth, W., Döbeli, H., Gentz, R. & Stüber, D. Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. <em>Nat. Biotechnol.</em> <b>6</b>, 1321–1325 (1988).</li>
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<li>Domeradzka, N. E., Werten, M. W., Wolf, F. A. and de Vries, R. Protein cross-linking tools for the construction of nanomaterials. <i>Curr Opin Biotechnol.</i> <b>39</b> 61-67, doi:10.1016/j.copbio.2016.01.003 (2016).</li>
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<li>Walper, S. A., Turner, K. B. and Medintz, I. L. Enzymatic bioconjugation of nanoparticles: developing specificity and control. <i>Curr Opin Biotechnol.</i> <b>34</b> 232-241, doi:10.1016/j.copbio.2015.04.003 (2015).</li>
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<li>Reddington, S. C. and Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. <i>Curr Opin Chem Biol.</i> <b>29</b> 94-99, doi:10.1016/j.cbpa.2015.10.002 (2015).</li>
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<li>Li, L., Fierer, J. O., Rapoport, T. A. and Howarth, M. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag. <i>J. Mol. Biol.</i> <b>426</b> 309–317 (2014).</li>
 
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Latest revision as of 03:30, 18 October 2018

Improved Part

Abstract

The UNSW iGEM team designed a new IaaH encoding part for improved functionality of the indole-3-aectamide hydrolase (IaaH) enzyme. Improving on the BBa_K89001 part submitted by the 2015 NUDT_China team, a HisTag and GSG linker were added to the N-terminus of the enzyme, and a SpyTag and GSG linker was added to the C-terminus. Following these additions, the team was able to successfully express and purify the new part utilising the HisTag's affinity for nickel ions to purify the enzyme using IMAC purification. We also demonstrated the ability of IaaH with SpyTag to covalently bind with SpyCatcher proteins through SDS-PAGE. More details can be found on the improved part's registry page at BBa_K2710005. Through these improvements, we have expanded the utility of this part twofold. First, the protein can be easily purified from cell lysates, and secondly, it now includes an irreversible attachment mechanism, robust over a range of conditions.

The Enzyme

Indole-3-acetamide hydrolase (IaaH) is an enzyme involved in the biosynthesis of indole-3-aectic acid. IaaH originating from Alcaligenes sp. strain HPC127114 was fused with a SpyTag and a hexahistidine tag (HisTag)1.

The auxin indole-3-acetic acid, is a plant hormone involved in the regulation of plant growth and development2. Indole-3-aecetic acid can be synthesised via the indole-3-acetamide pathway, which converts tryptophan to indole-3-aectic acid in a two-step enzymatic pathway3 (Figure 1). The flavoprotein tryptophan 2-monooxygenase (IaaM) catalyses the oxidative decarboxylation of tryptophan to indole-3-acetamide in the first, rate limiting step of the pathway4. Subsequently, the enzyme indole-3-acetamide hydrolase (IaaH) converts indole-3-acetamide to indole-3-aectic acid1.


Figure 1: The indole-3-acetamide pathway for indole-3-aecetic acid biosynthesis.


Improvements

The original part was improved by two key additions; a SpyTag and a HisTag.

The SpyTag forms one component of the SpyTag/SpyCatcher system, which enables covalent attachment of two proteins6. The SpyTag and SpyCatcher system was created by cleaving the CnaB2 domain of the fibronectin-binding protein FbaB derived from Streptococcus pyogenes to form a thirteen residue SpyTag peptide and a 116-residue SpyCatcher peptide6. The SpyTag (1.1 kDa) and SpyCatcher (12 kDa) form an irreversible intramolecular isopeptide bond between Asp117 on SpyTag and Lys31 on SpyCatcher6, spontaneously and specifically binding to each other so that they can be used as attachment mechanisms to create new, self-assembling protein arrangements6 (Figure 2).

It is particularly useful because neither component needs to be at the C or N terminus7, and the effect on the attached protein’s activity appears to be negligible8. It also reported as useful in a variety of reaction conditions, with Howarth showing that the SpyTag/SpyCatcher “had a high yield...required only simple mixing (and) tolerated diverse conditions (pH, buffer components and temperature)”9.

Figure 2: A spontaneous isopeptide bond forms between SpyTag and SpyCatcher. Image created using PDB ID: 4MLS10


A HisTag (six consecutive histidine residues, also known as a hexahistidine tag) was added to IaaH to enable purification, utilising the affinity of the HisTag for nickel ions for Immobilised Metal Affinity Chromatography purification5.


Experimental Characterisation

The improved part was validated by sequence verification and a diagnostic gel. Following this, the original and improved part were cloned into the expression vector pET19B and successfully expressed in E. coli T7 Express cells (NEB). The HisTag and SpyTag improvements were experimentally characterised through SDS-PAGE analysis of IMAC purifications and attachment to SpyCatcher fusion proteins. Refer to the BBa_K2710005 registry page for details of the experimental characterisation.

Through these improvements, we have expanded the utility of this part twofold. First, the protein can be easily purified from cell lysates, and secondly, it now includes an irreversible attachment mechanism, robust over a range of conditions.

Furthermore, as wild-type gPFD polymerises to form filamentous structures, Transmission Electron Microscopy (TEM) was performed on gPFD-SpyC attached to IaaH-SpyT to investigate the effect of the attachment of the enzyme on filament formation. Attachment via the SpyTag/SpyCatcher mechanism appears to distort the filaments (Figure 3).


Figure 3: TEM demonstrates that gPFD and gPFD mutants are able to assemble and form filaments. A: Wild-type gPFD filaments. B: gPFD-SpyC filaments, indicating that gPFD is able to form filaments when fused to a SpyCatcher. C: gPFD-SpyC reacted with IaaH-SpyT. Clumps of filaments, or curled filaments were observed.

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