Team:Michigan/Project

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<p> This year we set out to characterize and submit a part capable of secretion based single-chain variable fragment antibody protein purification. Utilizing osmotically inducible protein Y (OsmY)in the RFC25 protein fusion standard, we have created a construct capable of secreting a protein of interest from E. coli. Once secreted, our construct allows for simple cleavage and purification using the His-tag and TEV protease sites found between OsmY and the gene of interest. To characterize the construct, we plan to use a number of different proteins.  This will not only begin to assess what classes of proteins are capable of being purified and secreted by our construct, but will also allow us to assess the practicality of our construct in industry-level production of proteins.  We plan to purify antibodies against Salmonella protein Dihydroxyacid Dehydratase with our construct and ultimately compare the efficacy of our purified proteins to those of more traditional purification methods.<p>  
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<p> We believe that our protein purification construct will be remarkably useful for purifying proteins and could significantly decrease the cost of antibody production, as proteins of interest are secreted from the cell, decreasing the number of steps required to purify the protein significantly.  Future teams will be able to insert any protein of interest into the construct and secrete the protein into the cytoplasm of the cell, simplifying the purification process.  OsmY is an exceptionally useful protein for secretion based purification and our construct allows the iGEM community a modular vector for expression and purification of proteins. <p>
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<h1><font size="10" <em class = "colored">About our Project: OsmY</em></font></h1></p><font size="3"><font color="#191970"> 2014 Michigan Synthetic Biology Team </font></font color></p>
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    <p>Many important pharmaceutical protein, such as antibodies and insulin, are derived from mammalian organisms. These mammalian cells have organelles that isolate a special environment for proteins to fold correctly and form disulfide bonds, such as the endoplasmic reticulum. Yet, mammalian cells take a long, difficult time to grow  and are not ideal for industrial production of pharmaceutical proteins. Fast growing organism such as <i>E.coli</i> presented us with the grand advantage of producing and purifying large amounts of protein in a short amount of time but prokaryotes don’t have the organelles capable of properly folding pharmaceutical proteins.
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    </p><br><p>
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    Disulfide bonds occur naturally in <i>E.coli</i> in the periplasm. The periplasm has an oxidizing environment, similarly to the endoplasmic reticulum, that allows disulfide bond formation. If we drive a mammalian protein through the periplasm of an E.coli cell it will able to fold properly by disulfide bond formation.
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</p><br><p>
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    Antibodies have become paramount in biotechnology and medicine. Table 1 ranks the companies with the highest sales in March 2014; as we can see, 3 companies out of the top 10 sell antibody fragments. This reveals the impact of purification system improvement.
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    </p><br><p>
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    In our project, we've purified antibody fragments by tagging a secretion protein, OsmY, to an antibody scFv (single chain fragment variable) and over-expressing the construct in <i>E. coli</i>. To demonstrate the importance of our study we've chosen an antibody specific to DADH, a Salmonella protein. Salmonella is responsible for 23,000 hospitalizations in the US each year, highlighting the need for large quantities of inexpensive sensors. To further demonstrate the value of our part we've compared our secretion system with industrially used secretion system that use the secretion tag pelB. </p><p></p>
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<p> <font style="position:absolute;top:430px;left:250px"> Table 1 </font></p>
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<p> <font size="1" style="position:absolute;top:660px;left:500px"> "List of Therapeutic Monoclonal Antibodies." Wikipedia. Wikimedia Foundation, 13 Oct. 2014. Web. 14 Oct. 2014.
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"Salmonella." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 28 Aug. 2014. Web. 14 Oct. 2014.</p>
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{{MichiganMainScript}}

Latest revision as of 22:56, 14 October 2014

<! -- Michigan Synthetic Biology Team -->

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About our Project: OsmY

2014 Michigan Synthetic Biology Team

Many important pharmaceutical protein, such as antibodies and insulin, are derived from mammalian organisms. These mammalian cells have organelles that isolate a special environment for proteins to fold correctly and form disulfide bonds, such as the endoplasmic reticulum. Yet, mammalian cells take a long, difficult time to grow and are not ideal for industrial production of pharmaceutical proteins. Fast growing organism such as E.coli presented us with the grand advantage of producing and purifying large amounts of protein in a short amount of time but prokaryotes don’t have the organelles capable of properly folding pharmaceutical proteins.


Disulfide bonds occur naturally in E.coli in the periplasm. The periplasm has an oxidizing environment, similarly to the endoplasmic reticulum, that allows disulfide bond formation. If we drive a mammalian protein through the periplasm of an E.coli cell it will able to fold properly by disulfide bond formation.


Antibodies have become paramount in biotechnology and medicine. Table 1 ranks the companies with the highest sales in March 2014; as we can see, 3 companies out of the top 10 sell antibody fragments. This reveals the impact of purification system improvement.


In our project, we've purified antibody fragments by tagging a secretion protein, OsmY, to an antibody scFv (single chain fragment variable) and over-expressing the construct in E. coli. To demonstrate the importance of our study we've chosen an antibody specific to DADH, a Salmonella protein. Salmonella is responsible for 23,000 hospitalizations in the US each year, highlighting the need for large quantities of inexpensive sensors. To further demonstrate the value of our part we've compared our secretion system with industrially used secretion system that use the secretion tag pelB.

Table 1

"List of Therapeutic Monoclonal Antibodies." Wikipedia. Wikimedia Foundation, 13 Oct. 2014. Web. 14 Oct. 2014. "Salmonella." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 28 Aug. 2014. Web. 14 Oct. 2014.

Retrieved from "http://2014.igem.org/Team:Michigan/Project"