Difference between revisions of "Team:UCLA/Project/Protein Cages"

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<h2>Protein Cages</h2>
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          <h1 align="middle" style="position:relative;top:0%;text-decoration:none;font-family:helvetica;font-size:150%;background-color:#0A64A4;">BACKGROUND</h1>
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          <h1 align="middle" style="position:relative;top:0%;text-decoration:none;font-family:helvetica;font-size:150%;background-color:#0A64A4;">RESULTS</h1>
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          <h1 align="middle" style="position:relative;top:0%;text-decoration:none;font-family:helvetica;font-size:150%;background-color:#FFDE00;">BIOBRICKS</h1>
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<h1>Protein Cages</h1>
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<h2>Background</h2>
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<h4>Abstract</h4>
 
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Proteins naturally have evolved to self-assemble into supramolecular structures often playing a role in complex cellular functions. These supramolecular structures are built mostly by copies of the same protein subunit and are arranged in a symmetric fashion through non-covalent bonding. Though we see these in nature through specific proteins, what if we were able to engineer our own protein subunits and create complex structures for our own applications?
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The goal of our project is to modify a protein cage such that disassembly can be induced by thrombin protease.  Our protein cage is a previously-created synthetic cage composed of 12 subunits that form a tetrahedron. By creating mutant protein cages with inserted thrombin cleavage sites, disassembly could be induced by treatment with thrombin protease.  We designed 13 mutant protein cages and successfully cloned our top candidate based on selection criteria.  We were then able to successfully express and affinity purify both the native protein cage and the mutant protein cage.  However, complications with protein aggregation occurred, and upon assaying for protein cage assembly using dynamic light scattering, we found that our cages did not assemble at the expected size of 16nm.  Future directions will be focused on improving expression conditions for proper cage assembly, as well as on functionalizing the interior/exterior.
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<h4>Introduction</h4>
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<p>
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The seed project for next year involves the controlled disassembly of protein cages in response to thrombin protease.  What is a protein cage?  A protein cage is a type of supramolecular assembly, where multiple protein subunits interact non-covalently in order to form a higher-order structure. Classic examples of protein assemblies include viral capsids, ferritins, and clathrin coats1.  The cage that we are working with is a previously-designed synthetic protein cage2–4 (Figure 1), created by one of our advisors, Todd Yeates.  To make a subunit of the cage, two distinct, naturally oligomerizing proteins were selected—a trimer, bromoperoxidase, and a dimer, M1 matrix protein.  Each were fused with a linker to form a 109.5 degree angle.  This angle is crucial in that it allows 12 subunits to come together, in order to create a cage that has tetrahedral symmetry2.
 +
</p>
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<p>
 +
The goal of our project is to modify this protein cage such that disassembly can be induced by thrombin protease.  Proteases are enzymes that cleave at specific amino acid sequences; therefore, if our cage contained the sequence shown here, it can be disassembled by thrombin-induced cleavage.  This would have potential applications such as controlled release of loaded drugs/imaging molecules from inside the cage.  So why did we pick thrombin to work with? Thrombin was specifically chosen due to it being relatively well-studied, and because of its role in the coagulation cascade5, which contributes to heart disease and stroke the first and fifth leading causes of death in the United States, respectively7.
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A proven way to form these supramolecular structures is to engineer a self-assembling protein molecule as a fusion of two different, naturally oligomeric protein domains. In order to get a pre-defined, homogenous structure, the two components must be fused in a way that follows specific angular rules. To satisfy this requirement, the two proteins fused are required to have terminal alpha helices and are attached by an alpha helix preferring molecule. This allows the fused protein subunit to be sturdy without variance in its bond angles and allows us to predict with more accuracy on how the structure is going to form. This technique has worked and the predicted cage-like structure was achieved through a 12 subunit assembly.
 
  
  
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What can we do with such a cage? The most obvious application that comes to mind is drug delivery. We might be able to engineer a protease induced cleavage cite onto our cage making it an efficient delivery system where the cage breaks open the moment it comes in contact with its inducer and releases the drug. This technology can even lead to formation of protein cages made up of human proteins with non-immunogenic properties. Such a cage would have an immense impact in field of drug delivery and in the field of medicine as a whole.
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<h2>Results</h2>
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Using ICA, we have generated 10 silk constructs. These include constructs of pure MaSp2 ranging from 3-15 mers, pure MaSp1 of 9 and 12-mers and 12-mers of MaSp1/2 hybrids in 3 different ratios.  
 
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<h4>Future Directions</h4>
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<p>
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For future directions, the remaining mutants will be cloned, and the expression conditions will be optimized in order to obtain properly folded cages.  This will be assayed using techniques such as circular dichroism and dynamic light scattering.  Cages that fold properly will then be assayed for thrombin cleavage.  At which point, it would be possible to load the interior of the cage with drug/imaging molecules for release, or functionalize the exterior of the cage for specific receptor interactions.  Though the protein cage project is in a nascent phase, we hope to explore more possibilities that may shed light on using them for future applications in medicine.
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<h4>Achievements</h4>
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<ol>
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<li>
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Used Pymol software to model our protein cage and design 13 different protein cage mutants
 +
</li>
 +
<li>
 +
Cloned the top protein cage mutant based on our selection criteria
 +
</li>
 +
<li>
 +
Verified expression and affinity purification the native protein cage using SDS-PAGE
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</li>
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</ol>
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<h2>List of Biobricks</h2>
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<ul>
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<li>
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Protein Cage Mutant 10              BBa_K1763421
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<h2>References</h2>
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<p>
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1.  Goodsell, D. S. & Olson, A. J. Structural Symmetry and Protein Function. Annu. Rev. Biophys. Biomol. Struct. 105–53 (2000).
 +
</p>
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<p>
 +
2.  Padilla, J. E., Colovos, C. & Yeates, T. O. Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments. Proc. Natl. Acad. Sci. U. S. A. 98, 2217–2221 (2001).
 +
</p>
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<p>
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3.  Lai, Y.-T., Cascio, D. & Yeates, T. O. Structure of a 16-nm Cage Designed by Using Protein Oligomers. Science (80-. ). 336, 1129–1129 (2012).
 +
</p>
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<p>
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4.  Lai, Y.-T. & Al, E. Structure and Flexibility of Nanoscale Protein Cages Designed by Symmetric Self-Assembly. J Am Chem Soc 135, 7738–7743 (2013).
 +
</p>
 +
<p>
 +
5.  Adams, R. L. C. & Bird, R. J. Review article : Coagulation cascade and therapeutics update : Relevance to nephrology . Part 1 : Overview of coagulation , thrombophilias and history of anticoagulants. 462–470 (2009). doi:10.1111/j.1440-1797.2009.01128.x
 +
</p>
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<p>
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6.  Arzen, D., Co-advisor, R. P. & Ljubljana, D. K. Dynamic light scattering and application to proteins in solutions. (2010).
 +
</p>
 +
<p>
 +
7.  1. Goodsell, D. S. & Olson, A. J. Structural Symmetry and Protein Function. Annu. Rev. Biophys. Biomol. Struct. 105–53 (2000).
 +
2. Padilla, J. E., Colovos, C. & Yeates, T. O. Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments. Proc. Natl. Acad. Sci. U. S. A. 98, 2217–2221 (2001).
 +
3. Lai, Y.-T., Cascio, D. & Yeates, T. O. Structure of a 16-nm Cage Designed by Using Protein Oligomers. Science (80-. ). 336, 1129–1129 (2012).
 +
4. Lai, Y.-T. & Al, E. Structure and Flexibility of Nanoscale Protein Cages Designed by Symmetric Self-Assembly. J Am Chem Soc 135, 7738–7743 (2013).
 +
5. Adams, R. L. C. & Bird, R. J. Review article : Coagulation cascade and therapeutics update : Relevance to nephrology . Part 1 : Overview of coagulation , thrombophilias and history of anticoagulants. 462–470 (2009). doi:10.1111/j.1440-1797.2009.01128.x
 +
6. Arzen, D., Co-advisor, R. P. & Ljubljana, D. K. Dynamic light scattering and application to proteins in solutions. (2010).
 +
7.  http://www.cdc.gov/nchs/fastats/leading-causes-of-death.htm
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<h2>FIGURES: I DON'T KNOW WHERE TO PUT THEM</h2>
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<figcaption  style="margin: auto; width: 500px;">
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<b>Figure 1:</b>  Structure of a designed protein cage.  Geometric models illustrate the design principle of fusing two distinct, naturally oligomerizing proteins together in a specific geometry.  Twelve subunits combine to form a cage with tetrahedral symmetry.  Figure is adapted from Lai, Y.-T., Cascio, D. & Yeates, T. O. Structure of a 16-nm Cage Designed by Using Protein Oligomers. Science (80-. ). 336, 1129–1129 (2012).
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<figure style= "margin: 10px; float: right;"><img width="300px" src= "https://static.igem.org/mediawiki/2015/6/63/Fig_2_ICA_Cloning.png" />
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<figcaption  style="margin: auto; width: 300px;">
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<b>Fig 2:</b>Downstream Cloning after Iterative Capped Assembly. After elution from the beads, the ICA constructs are amplified using PCR primers that anneal to the initiator and the terminator. These primer binding sites are unique in the construct, and can be found nowhere else in the sequence. The only constructs that are amplified are those that have the initiator and terminator. All other constructs, while present, are excluded from amplification. After amplification, the construct can be cloned into a vector using traditional techniques.
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<figure style= "margin: 10px; float: left;"><img width="400px" src= "https://static.igem.org/mediawiki/2015/0/04/Fig_4_Accessory_Oligos.png" />
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<figcaption  style="margin: auto; width: 400px;">
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<b>Fig 4:</b>Schematic of examples of  initiator (a), terminator (b), and capping oligos (c), used in our ICA project. The cap shown has the B-type sticky end.
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<figure style= "margin: 10px; float: right;"><img width="400px" src= "https://static.igem.org/mediawiki/2015/1/12/Fig_5_Sticky_Ends.png" />
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<figcaption  style="margin: auto; width: 400px;">
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<b>Fig 5:</b>Schematic of the three types of sticky ends we designed for ICA. Sticky end A is 5’-AGTT-3’. Sticky end B is 5’-TGTC-3’. Sticky end C is 5’-CGTG-3’. An assembled 3-mer construct AB+BC+CA is shown as an example of how these biobricks would be used.
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<figure style= "margin: 10px; float: left;"><img width="600px" src= "https://static.igem.org/mediawiki/2015/c/ca/9_16_2015_UCLA_ICA_FINAL.jpg" />
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<b>Fig 6:</b>Gel image of constructs we created this summer using ICA. pSB1C3 Plasmids containing the sequence verified construct were digested using XbaI and PstI. Results were run on 1% TAE gel. The expected band size for the pSB1C3 is ~2070. Expected sizes for inserts fragments are indicated on the right hand side.
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Revision as of 19:17, 18 September 2015

iGEM UCLA




BACKGROUND

METHODOLOGY

RESULTS

BIOBRICKS























Protein Cages

Background

Abstract

The goal of our project is to modify a protein cage such that disassembly can be induced by thrombin protease. Our protein cage is a previously-created synthetic cage composed of 12 subunits that form a tetrahedron. By creating mutant protein cages with inserted thrombin cleavage sites, disassembly could be induced by treatment with thrombin protease. We designed 13 mutant protein cages and successfully cloned our top candidate based on selection criteria. We were then able to successfully express and affinity purify both the native protein cage and the mutant protein cage. However, complications with protein aggregation occurred, and upon assaying for protein cage assembly using dynamic light scattering, we found that our cages did not assemble at the expected size of 16nm. Future directions will be focused on improving expression conditions for proper cage assembly, as well as on functionalizing the interior/exterior.

Introduction

The seed project for next year involves the controlled disassembly of protein cages in response to thrombin protease. What is a protein cage? A protein cage is a type of supramolecular assembly, where multiple protein subunits interact non-covalently in order to form a higher-order structure. Classic examples of protein assemblies include viral capsids, ferritins, and clathrin coats1. The cage that we are working with is a previously-designed synthetic protein cage2–4 (Figure 1), created by one of our advisors, Todd Yeates. To make a subunit of the cage, two distinct, naturally oligomerizing proteins were selected—a trimer, bromoperoxidase, and a dimer, M1 matrix protein. Each were fused with a linker to form a 109.5 degree angle. This angle is crucial in that it allows 12 subunits to come together, in order to create a cage that has tetrahedral symmetry2.

The goal of our project is to modify this protein cage such that disassembly can be induced by thrombin protease. Proteases are enzymes that cleave at specific amino acid sequences; therefore, if our cage contained the sequence shown here, it can be disassembled by thrombin-induced cleavage. This would have potential applications such as controlled release of loaded drugs/imaging molecules from inside the cage. So why did we pick thrombin to work with? Thrombin was specifically chosen due to it being relatively well-studied, and because of its role in the coagulation cascade5, which contributes to heart disease and stroke the first and fifth leading causes of death in the United States, respectively7.

Results

Using ICA, we have generated 10 silk constructs. These include constructs of pure MaSp2 ranging from 3-15 mers, pure MaSp1 of 9 and 12-mers and 12-mers of MaSp1/2 hybrids in 3 different ratios.

Future Directions

For future directions, the remaining mutants will be cloned, and the expression conditions will be optimized in order to obtain properly folded cages. This will be assayed using techniques such as circular dichroism and dynamic light scattering. Cages that fold properly will then be assayed for thrombin cleavage. At which point, it would be possible to load the interior of the cage with drug/imaging molecules for release, or functionalize the exterior of the cage for specific receptor interactions. Though the protein cage project is in a nascent phase, we hope to explore more possibilities that may shed light on using them for future applications in medicine.

Achievements

  1. Used Pymol software to model our protein cage and design 13 different protein cage mutants
  2. Cloned the top protein cage mutant based on our selection criteria
  3. Verified expression and affinity purification the native protein cage using SDS-PAGE

List of Biobricks

  • Protein Cage Mutant 10 BBa_K1763421

References

1. Goodsell, D. S. & Olson, A. J. Structural Symmetry and Protein Function. Annu. Rev. Biophys. Biomol. Struct. 105–53 (2000).

2. Padilla, J. E., Colovos, C. & Yeates, T. O. Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments. Proc. Natl. Acad. Sci. U. S. A. 98, 2217–2221 (2001).

3. Lai, Y.-T., Cascio, D. & Yeates, T. O. Structure of a 16-nm Cage Designed by Using Protein Oligomers. Science (80-. ). 336, 1129–1129 (2012).

4. Lai, Y.-T. & Al, E. Structure and Flexibility of Nanoscale Protein Cages Designed by Symmetric Self-Assembly. J Am Chem Soc 135, 7738–7743 (2013).

5. Adams, R. L. C. & Bird, R. J. Review article : Coagulation cascade and therapeutics update : Relevance to nephrology . Part 1 : Overview of coagulation , thrombophilias and history of anticoagulants. 462–470 (2009). doi:10.1111/j.1440-1797.2009.01128.x

6. Arzen, D., Co-advisor, R. P. & Ljubljana, D. K. Dynamic light scattering and application to proteins in solutions. (2010).

7. 1. Goodsell, D. S. & Olson, A. J. Structural Symmetry and Protein Function. Annu. Rev. Biophys. Biomol. Struct. 105–53 (2000). 2. Padilla, J. E., Colovos, C. & Yeates, T. O. Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments. Proc. Natl. Acad. Sci. U. S. A. 98, 2217–2221 (2001). 3. Lai, Y.-T., Cascio, D. & Yeates, T. O. Structure of a 16-nm Cage Designed by Using Protein Oligomers. Science (80-. ). 336, 1129–1129 (2012). 4. Lai, Y.-T. & Al, E. Structure and Flexibility of Nanoscale Protein Cages Designed by Symmetric Self-Assembly. J Am Chem Soc 135, 7738–7743 (2013). 5. Adams, R. L. C. & Bird, R. J. Review article : Coagulation cascade and therapeutics update : Relevance to nephrology . Part 1 : Overview of coagulation , thrombophilias and history of anticoagulants. 462–470 (2009). doi:10.1111/j.1440-1797.2009.01128.x 6. Arzen, D., Co-advisor, R. P. & Ljubljana, D. K. Dynamic light scattering and application to proteins in solutions. (2010). 7. http://www.cdc.gov/nchs/fastats/leading-causes-of-death.htm

FIGURES: I DON'T KNOW WHERE TO PUT THEM

Figure 1: Structure of a designed protein cage. Geometric models illustrate the design principle of fusing two distinct, naturally oligomerizing proteins together in a specific geometry. Twelve subunits combine to form a cage with tetrahedral symmetry. Figure is adapted from Lai, Y.-T., Cascio, D. & Yeates, T. O. Structure of a 16-nm Cage Designed by Using Protein Oligomers. Science (80-. ). 336, 1129–1129 (2012).
Fig 2:Downstream Cloning after Iterative Capped Assembly. After elution from the beads, the ICA constructs are amplified using PCR primers that anneal to the initiator and the terminator. These primer binding sites are unique in the construct, and can be found nowhere else in the sequence. The only constructs that are amplified are those that have the initiator and terminator. All other constructs, while present, are excluded from amplification. After amplification, the construct can be cloned into a vector using traditional techniques.
Fig 4:Schematic of examples of initiator (a), terminator (b), and capping oligos (c), used in our ICA project. The cap shown has the B-type sticky end.
Fig 5:Schematic of the three types of sticky ends we designed for ICA. Sticky end A is 5’-AGTT-3’. Sticky end B is 5’-TGTC-3’. Sticky end C is 5’-CGTG-3’. An assembled 3-mer construct AB+BC+CA is shown as an example of how these biobricks would be used.
Fig 6:Gel image of constructs we created this summer using ICA. pSB1C3 Plasmids containing the sequence verified construct were digested using XbaI and PstI. Results were run on 1% TAE gel. The expected band size for the pSB1C3 is ~2070. Expected sizes for inserts fragments are indicated on the right hand side.