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{{NortheasternU-Boston}}
 
{{NortheasternU-Boston}}
 
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        </html>{{NortheasternU-Boston-Sidebar}}<html>
  
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<h1>Cell-Free Protein Expression to Better Distribute Medicine</h1>
+
<div class="header">
−
 
+
            <h1>Project Description</h1>
−
<p>Our Team project this year is focusing on the characterization and development of cell-free systems for protein expression in the hopes of increasing the ability of these systems to produce medicinal biologics for areas without advanced medical infrastructure. </p>
+
          </div>
 +
          <div class="content-text">
 +
<h3>Inspiration</h3>
 +
<p>
 +
Our team wanted to work with a synthetic biology system aimed at improving
 +
access to medicine in areas of the world lacking in strong medical and shipping
 +
infrastructure. While searching for ideas in this sphere we came across the notion
 +
of Cell-free expression reactions on a shelf stable paper substrate
 +
(Pardee, et al 2014, Pardee, et al. 2016). We were struck by the notion of
 +
circumventing the cold-chain and excited by the idea of building our own cell-free
 +
expression systems to produce medications for the developing world. As we sunk our
 +
teeth into the literature we realized that it would be difficult to find a realistic
 +
candidate for a medicine which was:</p>
 +
<ol>
 +
  <li>Limited by cold-chain shipping and storage</li>
 +
  <li>A simple enough compound to be produced in a cell free context</li>
 +
  <li>Would have important medical impact for the developing world</li>
 +
</ol>
 +
<p>
 +
We decided to focus our attention
 +
on the problem of complexity and decided that rather than focus on the medicinal
 +
aspect of the problem we would focus our efforts instead on characterizing and
 +
improving cell-free reaction platforms in order to produce more complex biologics.
 +
Specifically we wanted to focus on post-translational modifications. Expanding
 +
cell-free reactions with post-translational modifications for important functions
 +
like glycosylation or cleavage and subsequent rearrangement to occur; offering
 +
the chance to produce complex biologics like antibodies or insulin in a cell-free
 +
context (Knorre et al. 2009).
 +
</p>
  
 +
<h3>Model System</h3>
 
<p>
 
<p>
−
Our team this year became interested in diseases that were treatable but undertreated, especially in the healthcare space of diseases that had effective treatments but where access to these treatments was the limiting factor leading to unnecessary suffering. Specifically our group became drawn to sub-Saharan Africa where infectious diseases cause roughly a third of all deaths [1]. Many of these diseases are treatable but access to the treatment  is limited. One key limiting factor is the availability of cold-chain distribution. Many medicinal biologics like vaccines and recombinant proteins are rapidly denatured and destroyed when not refrigerated and in order for them to maintain medical usability they need to be manufactured, shipped, and stored at cool temperatures. In areas without consistent electricity or reliable roads for refrigerated transport this is prohibitively difficult.  
+
The space of post-translational modifications is huge, and so the team needed to
 +
focus on a specific set of molecules and a set of post-translational modifications.
 +
We decided to work with antimicrobial peptides (AMPs) -- short peptides which exist
 +
as part of the immune system in many kinds of derived eukaryotes (Izadpanah et al. 2005).
 +
These were an ideal choice of model for several reasons.
 +
Groups had already worked with AMPs in a cell-free context using reaction preparations
 +
that were feasible for us to recapitulate in our lab and there is a large amount of
 +
diverse research on AMPs in general (Hancock et al. 2006, Pardee et al. 2016, Wu et al. 2014).
 +
There is a broad database of AMPs from a myriad of organisms which target broadly
 +
and specifically organisms from <i>E. coli</i> to <i>P. falciparum</i> (Wang et al. 2016).The
 +
diversity of this class of peptide made it attractive as a model because we could
 +
investigate factors like length, class of AMP, and predicted or known secondary
 +
structure. Producing AMPs across a complexity spectrum would give us some hint
 +
as to the capability of our cell-free system. AMPs also offered a key opportunity
 +
as a model in that many of them do not begin with a methionine as part of their
 +
peptide sequence. In our E. coli extract based system of cell-free expression we
 +
needed our expressed peptides to begin with a methionine start codon.
 +
For AMPs which do not have a canonical N-terminal methionine this residue would have to be removed in a post-translational
 +
reaction. We searched the literature for a way to achieve this and found work
 +
by Liao et al. (2004) working on optimizing mutants of methionine aminopeptidase
 +
isolated from <i>E. coli</i>. They presented findings of a GTG triple mutant methionine
 +
aminopeptidase with improved ability to cleave N-terminal methionines from peptides
 +
with bulky penultimate N-terminal residues. We now had our model system:
 +
Pick AMPs across a complexity gradient, with and without canonical N-terminal
 +
methionines and then test these for function against bacteria with and without
 +
the presence of methionine aminopeptidase.
 
</p>
 
</p>
  
−
<h5>
+
<h3>Cell Free Reactions</h3>
−
One potential way of circumventing the cold chain is to use freeze-dried cell-free systems to produce medicinal biologics at the point of care.
+
−
<h5>
+
−
 
+
 
<p>
 
<p>
−
These systems are shelf-stable at room temperature and are economically viable [2]. Our project will use a variety of anti-microbial peptides of varying complexity in order to demonstrate the capabilities and limitations of our system in producing proteins of measurable medical impact. We hope to elucidate the various limiting factors of cell-free expression (protein size, secondary structure complexity, and post translational modification) and to improve the capability of cell-free expression to produce biologics of greater complexity and medical relevance.  
+
Working with the limitations of our lab space and set up, and with the idea in
 +
mind to create the most economically viable cell-free reactions possible we decided
 +
to work with a system based off of crude <i>E. coli</i> extract. We adapted methodologies
 +
from various sources to the tools that were available to us
 +
(Kwon et al. 2015, Pardee et al. 2016, Sun et al 2013). CF reactions were made by
 +
growing up T7 Express <i>E. coli</i> induced to express the T7 polymerase.
 +
Homogenizing these cells and collecting a supernatant allowed to undergo a run-off
 +
reaction to digest endogenous RNA and DNA and then the subsequent combination of
 +
this crude extract with the supplementary buffers, tRNAs, and other compounds found
 +
in Sun et al. 2013, according to their protocol. The resulting expression mixture
 +
was then aliquoted onto paper strips in microcentrifuge tubes, flash frozen, and
 +
freeze dried over 24 hours and was then ready for use.
 
</p>
 
</p>
  
 +
<h3>Citations</h3>
 +
<ul>
 +
  <li>
 +
    <p>
 +
Guangshun Wang, Xia Li, Zhe Wang; APD3: the antimicrobial peptide database as a
 +
tool for research and education, Nucleic Acids Research, Volume 44, Issue D1,
 +
4 January 2016, Pages D1087–D1093,
 +
<a href="https://doi.org/10.1093/nar/gkv1278" target="_blank">https://doi.org/10.1093/nar/gkv1278</a>
 +
    </p>
 +
  </li>
 +
  <li>
 +
    <p>
 +
Hancock, R. E. W., &amp; Sahl, H.-G. (2006). Antimicrobial and host-defense
 +
peptides as new anti-infective therapeutic strategies. 24, 1551. doi:10Wu.1038/nbt1267
 +
<a href="http://www.nature.com/nbt/journal/v24/n12/full/nbt1267.html" target="_blank">http://www.nature.com/nbt/journal/v24/n12/full/nbt1267.html</a>
 +
    </p>
 +
  </li>
 +
  <li>
 +
    <p>
 +
Izadpanah, A., Gallo, R.L., 2005. Antimicrobial peptides. J. Am. Acad. Dermatol.
 +
52, 381-390; quiz 391-392. doi:10.1016/j.jaad.2004.08.026
 +
<a href="https://www.ncbi.nlm.nih.gov/pubmed/15761415" target="_blank">https://www.ncbi.nlm.nih.gov/pubmed/15761415</a>
 +
    </p>
 +
  </li>
 +
  <li>
 +
    <p>
 +
Knorre, D.G., Kudryashova, N.V., Godovikova, T.S., 2009. Chemical and Functional
 +
Aspects of Posttranslational Modification of Proteins. Acta Naturae 1, 29–51.
 +
<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347534/" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347534/</a>
 +
    </p>
 +
  </li>
 +
  <li>
 +
    <p>
 +
Kwon, Y.-C., &amp; Jewett, M. C. (2015). High-throughput preparation methods of
 +
crude extract for robust cell-free protein synthesis. 5, 8663. doi:10.1038/srep08663
 +
<a href="https://www.nature.com/articles/srep08663#supplementary-information" target="_blank">https://www.nature.com/articles/srep08663#supplementary-information</a>
 +
    </p>
 +
  </li>
 +
  <li>
 +
    <p>
 +
Pardee, Keith, et al. “Paper-Based Synthetic Gene Networks.” Cell, vol. 159, no.
 +
4, Nov. 2014, pp. 940–54. www.cell.com, doi:10.1016/j.cell.2014.10.004.
 +
<a href="http://www.cell.com/abstract/S0092-8674(14)01291-4" target="_blank">http://www.cell.com/abstract/S0092-8674(14)01291-4</a>
 +
    </p>
 +
  </li>
 +
  <li>
 +
    <p>
 +
Pardee, K., Slomovic, S., Nguyen, P. Q., Lee, J. W., Donghia, N., Burrill, D., . . .
 +
Collins, J. J. (2016). Portable, On-Demand Biomolecular Manufacturing. Cell, 167(1),
 +
248-259.e212. doi:<a href="https://doi.org/10.1016/j.cell.2016.09.013" target="_blank">https://doi.org/10.1016/j.cell.2016.09.013</a>
 +
<a href="http://www.sciencedirect.com.ezproxy.neu.edu/science/article/pii/S0092867416312466" target="_blank">http://www.sciencedirect.com.ezproxy.neu.edu/science/article/pii/S0092867416312466</a>
 +
    </p>
 +
  </li>
 +
  <li>
 +
    <p>
 +
Liao, Y.-D., Jeng, J.-C., Wang, C.-F., Wang, S.-C., &amp; Chang, S.-T. (2004).
 +
Removal of N-terminal methionine from recombinant proteins by engineered
 +
E. coli methionine aminopeptidase. Protein Science : A Publication of the Protein Society,
 +
13(7), 1802-1810. doi:10.1110/ps.04679104
 +
<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2279930/" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2279930/</a>
 +
    </p>
 +
  </li>
 +
  <li>
 +
    <p>
 +
Wu, X., Wang, Z., Li, X., Fan, Y., He, G., Wan, Y., . . . Yang, L. (2014).
 +
In Vitro and In Vivo Activities of Antimicrobial Peptides Developed Using an
 +
Amino Acid-Based Activity Prediction Method. Antimicrobial Agents and Chemotherapy,
 +
58(9), 5342-5349. doi:10.1128/AAC.02823-14
 +
<a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135812/" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135812/</a>
 +
    </p>
 +
  </li>
 +
  <li>
 +
    <p>
 +
Sun, Z. Z., Hayes, C. A., Shin, J., Caschera, F., Murray, R. M., Noireaux, V. (2013).
 +
Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression
 +
System for Synthetic Biology. J. Vis. Exp. (79), e50762, doi:10.3791/50762
 +
<a href="https://www.jove.com/video/50762/protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free" target="_blank">https://www.jove.com/video/50762/protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free</a>
 +
    </p>
 +
  </li>
 +
</ul>
  
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<h5>Advice on writing your Project Description</h5>
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We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be consist, accurate and unambiguous in your achievements.
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Judges like to read your wiki and know exactly what you have achieved. This is how you should think about these sections; from the point of view of the judge evaluating you at the end of the year.
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Latest revision as of 01:39, 2 November 2017

Project Description

Inspiration

Our team wanted to work with a synthetic biology system aimed at improving access to medicine in areas of the world lacking in strong medical and shipping infrastructure. While searching for ideas in this sphere we came across the notion of Cell-free expression reactions on a shelf stable paper substrate (Pardee, et al 2014, Pardee, et al. 2016). We were struck by the notion of circumventing the cold-chain and excited by the idea of building our own cell-free expression systems to produce medications for the developing world. As we sunk our teeth into the literature we realized that it would be difficult to find a realistic candidate for a medicine which was:

  1. Limited by cold-chain shipping and storage
  2. A simple enough compound to be produced in a cell free context
  3. Would have important medical impact for the developing world

We decided to focus our attention on the problem of complexity and decided that rather than focus on the medicinal aspect of the problem we would focus our efforts instead on characterizing and improving cell-free reaction platforms in order to produce more complex biologics. Specifically we wanted to focus on post-translational modifications. Expanding cell-free reactions with post-translational modifications for important functions like glycosylation or cleavage and subsequent rearrangement to occur; offering the chance to produce complex biologics like antibodies or insulin in a cell-free context (Knorre et al. 2009).

Model System

The space of post-translational modifications is huge, and so the team needed to focus on a specific set of molecules and a set of post-translational modifications. We decided to work with antimicrobial peptides (AMPs) -- short peptides which exist as part of the immune system in many kinds of derived eukaryotes (Izadpanah et al. 2005). These were an ideal choice of model for several reasons. Groups had already worked with AMPs in a cell-free context using reaction preparations that were feasible for us to recapitulate in our lab and there is a large amount of diverse research on AMPs in general (Hancock et al. 2006, Pardee et al. 2016, Wu et al. 2014). There is a broad database of AMPs from a myriad of organisms which target broadly and specifically organisms from E. coli to P. falciparum (Wang et al. 2016).The diversity of this class of peptide made it attractive as a model because we could investigate factors like length, class of AMP, and predicted or known secondary structure. Producing AMPs across a complexity spectrum would give us some hint as to the capability of our cell-free system. AMPs also offered a key opportunity as a model in that many of them do not begin with a methionine as part of their peptide sequence. In our E. coli extract based system of cell-free expression we needed our expressed peptides to begin with a methionine start codon. For AMPs which do not have a canonical N-terminal methionine this residue would have to be removed in a post-translational reaction. We searched the literature for a way to achieve this and found work by Liao et al. (2004) working on optimizing mutants of methionine aminopeptidase isolated from E. coli. They presented findings of a GTG triple mutant methionine aminopeptidase with improved ability to cleave N-terminal methionines from peptides with bulky penultimate N-terminal residues. We now had our model system: Pick AMPs across a complexity gradient, with and without canonical N-terminal methionines and then test these for function against bacteria with and without the presence of methionine aminopeptidase.

Cell Free Reactions

Working with the limitations of our lab space and set up, and with the idea in mind to create the most economically viable cell-free reactions possible we decided to work with a system based off of crude E. coli extract. We adapted methodologies from various sources to the tools that were available to us (Kwon et al. 2015, Pardee et al. 2016, Sun et al 2013). CF reactions were made by growing up T7 Express E. coli induced to express the T7 polymerase. Homogenizing these cells and collecting a supernatant allowed to undergo a run-off reaction to digest endogenous RNA and DNA and then the subsequent combination of this crude extract with the supplementary buffers, tRNAs, and other compounds found in Sun et al. 2013, according to their protocol. The resulting expression mixture was then aliquoted onto paper strips in microcentrifuge tubes, flash frozen, and freeze dried over 24 hours and was then ready for use.

Citations

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