Team:TU Eindhoven/Background/Membrane Anchors

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                   <h2>SPAAC Reaction: Bio-Orthogonal Click Chemistry</h2>
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                   <h2>Membrane Anchors: CPX and INPNC (<a href='http://parts.igem.org/Part:BBa_K811005' target="_blank">BBa K811005</a>)</h2>
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                   <p>In order to functionalize bacterial membranes with polymers, two strategies can be followed: one can either engineer the bacteria in such a way that they produce the entire polymer, this strategy is further discussed under <a href="https://2014.igem.org/Team:TU_Eindhoven/ZAP">Zwitterionic Antifouling Protein</a>, or only produce an ‘anchor’ on which polymers can be reacted.  The latter strategy requires so-called bio-orthogonal chemistry; chemistry in which the two components are non-interacting (orthogonal) to the functionality presented in biological systems.  Furthermore, the reaction conditions have to be viable for cells; in water, at (near-) neutral pH, at temperatures ranging from 25 to 37°C and without any cytotoxic reagents or by-products. (Baskin & Bertozzi, 2007)
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                   <p>In order to click molecules over the entire cell surface, a protein to anchor the desired coating to the cell is needed. This protein has to be modified in such a way that it contains an azide which is displayed on the outside of the cell, where it can react with a DBCO-conjugate. Check also our <a href='https://2014.igem.org/Team:TU_Eindhoven/Background/SPAAC_Reaction'>SPAAC Reaction</a> Page for detailed information.</p>
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A common functional group used in bio-orthogonal reactions is the azide, which does not exist among or reacts with functional groups in biology and is both kinetically stable and thermodynamically high in energy to specific reactivity. Using the azide functional group, two components can be linked together inherently efficient, therefore those types of reactions are called click reactions. (Baskin & Bertozzi, 2007) Until now, three bio-orthogonal click reactions are known:</p>
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<ol style="color:white;margin-bottom:30px;">
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                  <h3>Membrane Anchor Protein CPX</h3>
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      <li><a href='#Staudinger'>Staudinger Ligation</a></li> 
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<p>CPX is a membrane protein designed for bacterial display. This membrane protein originates from OmpX. OmpX (Outer Membrane Protein x) is a small, monomeric β-barrel protein that is highly expressed and very useful for protein display applications in <i>E. coli</i>. CPX, or Circularly permutated OmpX, was developed as a bacterial display methodology for N- and C-terminal display. It is demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. OmpX possesses four extracellular loops, with loops 2 and 3 forming semi rigid β-sheets protruding from the cell surface. The native N- and C-termini were fused together with a GGSG linker, and the newly formed N- and C-termini reside on the cell surface. See <a href='#Fig1'>Figure 1</a> for a schematic overview of CPX and OmpX. [1]
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      <li><a href='#Copper'>Copper catalysed [3+2] azide-alkyne cycloaddition</a></li>
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      <li><a href='#Strain'>Strain promoted [3+2] azide-alkyne cycloaddition</a></li>
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<img id='Fig1' src="https://static.igem.org/mediawiki/2014/5/5e/TU_Eindhoven_OmpX_CPX.png" class="image_wrapper image_fr" width="1085">
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<p style="font-size:18px;color:#CCCCCC;">Figure 1. Structure of OmpX alongside a topological depiction of OmpX and CPX.</p>
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                  <h3>Membrane Anchor Protein INPNC</h3>
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The main advantage of these reactions is that they can be applied to in vivo incorporation of unnatural amino acids containing azides or alkynes during translation and expression of proteins. In this way, in theory all bio-orthogonal molecules, such as fluorescent or chemical reporters, can be ‘clicked’ on proteins. (Meldal & Tornoe, 2008)
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<p>In 2012 the iGEM team of Penn, University of Pennsylvania, created a generalized BioBrick surface display platform, the Ice Nucleation Protein N, C termini only (INPNC), BioBrick part: <a href='http://parts.igem.org/Part:BBa_K811005' target="_blank">BBa_K811005</a>.
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INPNC is a truncated Ice Nucleation Protein (INP, a protein which causes ice nucleation and formation but which is also used for its surface display properties). However, INP consists of a C-terminal region that is positioned on the outer membrane, as well as a central 8, 16 or 48 amino acid motif that is responsible for INP’s ice nucleation properties. These central amino acids motifs are not essential for INP’s surface displaying properties. Therefore INP was truncated, retaining only the N (179 aa) and C termini (49 aa) to create INPNC. The C-terminal domain is displayed on the cell surface, while the N-terminal domain remains in the outer membrane. Also check the <a href='https://2012.igem.org/Team:Penn'>Wiki Page</a> of Penn iGEM Team 2012. [2]
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<h3 id='Staudinger'>Staudinger Ligation</h3>
 
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                  <p>The Staudinger ligation is a modification of the classical Staudinger reaction, which implies the use of phosphines and azides. <NOBR>(<a href='#Fig1'>Figure 1</a>)</NOBR> The Staudinger ligation is arguably the first bio-orthogonal reaction and involves two fully abiotic functional groups and takes place at ambient temperature, in water and at neutral pH. (Baskin & Bertozzi, 2007) However, Vugts et al. showed that the Staudinger ligation is not bio-orthogonal and efficient enough in mice and that the slow reaction kinetics also severely restrict the applicability of the Staudinger ligation in vivo. (Vugts, et al., 2011)</p>
 
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<img id='Fig1' src="https://static.igem.org/mediawiki/2014/1/1d/TU_Eindhoven_Staudinger_ligation.png" class="image_wrapper image_fr" width="1085">
 
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<p style="font-size:18px;color:#CCCCCC;">Figure 1. A schematic overview of the Staudinger ligation.</p>
 
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<h3 id='Copper'>Copper Catalysed [3+2] Azide-Alkyne Cycloaddition (CuAAC)</h3>
 
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                  <p>The reaction often is referred to as ‘the click reaction’, is the extremely selective copper catalysed [3+2] cycloaddition of an azide and an alkyne (CuAAC). (<a href='#Fig2'>Figure 2</a>) CuAAC is a variant of the classical [3+2] cycloaddition discovered by Huisgen, in which copper serves as catalyst, creating a reaction that proceeds rapidly at room temperature. The copper is also the biggest disadvantage of this reaction, since this heavy metal is toxic to cells and organisms. (Meldal & Tornoe, 2008) Nowadays, several suitable Cu(I) ligands are available that minimize the cytotoxicity while maintaining or even further increasing the rate of CuAAC. Therefore, CuAAC has become a very feasible option for in vivo studies. (Yang, Li, & Chen, 2014)</p>
 
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<img id='Fig2' src="https://static.igem.org/mediawiki/2014/c/c2/TU_Eindhoven_CuAAC.png" class="image_wrapper image_fr" width="1085">
 
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<p style="font-size:18px;color:#CCCCCC;">Figure 2. A schematic overview of CuAAC.</p>
 
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<h3 id='Strain'>Strain Promoted [3+2] Azide-Alkyne Cycloaddition (SPAAC)</h3>
 
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                  <p>In order to avoid the disadvantages of copper, a copper free version of CuAAC called SPAAC has been designed. In SPAAC, instead of copper, ring strain is used to activate the alkyne. (<a href='#Fig3'>Figure 3</a>) Agard et al. showed that an appropriately derivatized cyclooctyne can efficiently and selectively react with azides in biological mixtures. (Agard, Prescher, & Bertozzi, 2004)</p>
 
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<img id='Fig3' src="https://static.igem.org/mediawiki/2014/3/3f/TU_Eindhoven_SPAAC.png" class="image_wrapper image_fr" width="1085">
 
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<p style="font-size:18px;color:#CCCCCC;">Figure 3. A schematic overview of SPAAC.</p>
 
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<p>Because of the efficiency and selectivity of SPAAC and the fact that no further reagents have to be added to the reaction mixture, SPAAC was chosen to further explore as a platform to functionalize bacterial membranes. Currently, the fastest cyclooctyne known is BARAC. However, this cyclooctyne has shown to be susceptible to Michael addition by thiols , which is not favourable when applying the system into a protein, since proteins contain lots of thiols (cysteines). Dibenzocycloocytne (DBCO) has a lower rate constant, but does not show susceptibility to Michael addition. (Debets, et al., 2014) Furthermore, there are already several DBCO-functionalized polymers and fluorophores commercially available. Therefore, the SPAAC reaction between DBCO-functionalized molecules, which will be reacted azide-functionalized amino acids that have been incorporated into the membrane of <i>E. coli</i>. (<a href='#Fig4'>Figure 4</a>)</p>
 
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<img id='Fig4' src="https://static.igem.org/mediawiki/2014/5/57/TU_Eindhoven_SPAAC_-_DBCO.png" class="image_wrapper image_fr" width="1085">
 
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<p style="font-size:18px;color:#CCCCCC;">Figure 4. A schematic overview of the bio-orthogonal click chemistry that will be conducted in our project: SPAAC using a DBCO-functionalized polymer A and an azide-functionalized amino acid B.</p>
 
<h4>Bibliography</h4>
<h4>Bibliography</h4>
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<p>Agard, N. J., Prescher, J. A., & Bertozzi, C. R. (2004). A strain-promoted [3+2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. Journal of the American Chemical Society, 126, 15046-15047.
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<p>[1] Rice, J. J.. "Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands." Protein Science 15.4 (2006): 825-836.
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Baskin, J. M., & Bertozzi, C. R. (2007). Bioorthogonal click chemistry: covalent labeling in living systems. QSAR & Combinatorial Science, 26(11-12), 1211 - 1219.
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[2] <a href="https://2012.igem.org/Team:Penn" target="_blank">Penn iGEM 2012</a>
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</p>
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Debets, M. F., Prins, J. S., Merkx, D., van Berkel, S. S., van Delft, F. L., van Hest, J. C., & Rutjes, F. P. (2014). Synthesis of DIBAC analogues with excellent SPAAC rate constants. Organic & Biomolecular Chemistry, 12, 5031-5037.
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Meldal, M., & Tornoe, C. W. (2008). Cu-catalyzed azide-alkyne cycloaddition. Chemical Reviews, 108, 2952-3015.
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Vugts, D. J., Vervoort, A., Stigter-van Walsum, M., Visser, G. W., Robillard, M. S., Versteegen, R. M., . . . van Dongen, G. A. (2011). Synthesis of phosphine and antibody-azide probes for in vivo Staudinger ligation in a pretargeted imaging and therapy approach. Bioconjugate Chemistry, 22, 2072-2081.
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Yang, M., Li, J., & Chen, P. R. (2014). Transition metal-mediated bioorthogonal protein chemistry in living cells. Chemical Society Reviews, 43(18), 6475-6660.
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Latest revision as of 00:09, 18 October 2014

iGEM Team TU Eindhoven 2014

iGEM Team TU Eindhoven 2014

Membrane Anchors: CPX and INPNC (BBa K811005)

In order to click molecules over the entire cell surface, a protein to anchor the desired coating to the cell is needed. This protein has to be modified in such a way that it contains an azide which is displayed on the outside of the cell, where it can react with a DBCO-conjugate. Check also our SPAAC Reaction Page for detailed information.

Membrane Anchor Protein CPX

CPX is a membrane protein designed for bacterial display. This membrane protein originates from OmpX. OmpX (Outer Membrane Protein x) is a small, monomeric β-barrel protein that is highly expressed and very useful for protein display applications in E. coli. CPX, or Circularly permutated OmpX, was developed as a bacterial display methodology for N- and C-terminal display. It is demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. OmpX possesses four extracellular loops, with loops 2 and 3 forming semi rigid β-sheets protruding from the cell surface. The native N- and C-termini were fused together with a GGSG linker, and the newly formed N- and C-termini reside on the cell surface. See Figure 1 for a schematic overview of CPX and OmpX. [1]

Figure 1. Structure of OmpX alongside a topological depiction of OmpX and CPX.

Membrane Anchor Protein INPNC

In 2012 the iGEM team of Penn, University of Pennsylvania, created a generalized BioBrick surface display platform, the Ice Nucleation Protein N, C termini only (INPNC), BioBrick part: BBa_K811005. INPNC is a truncated Ice Nucleation Protein (INP, a protein which causes ice nucleation and formation but which is also used for its surface display properties). However, INP consists of a C-terminal region that is positioned on the outer membrane, as well as a central 8, 16 or 48 amino acid motif that is responsible for INP’s ice nucleation properties. These central amino acids motifs are not essential for INP’s surface displaying properties. Therefore INP was truncated, retaining only the N (179 aa) and C termini (49 aa) to create INPNC. The C-terminal domain is displayed on the cell surface, while the N-terminal domain remains in the outer membrane. Also check the Wiki Page of Penn iGEM Team 2012. [2]

Bibliography

[1] Rice, J. J.. "Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands." Protein Science 15.4 (2006): 825-836.

[2] Penn iGEM 2012

iGEM Team TU Eindhoven 2014

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