Team:Melbourne/Project

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<h3>Synthesis approach</h3>
<h3>Synthesis approach</h3>
<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains,  disulfide bonds are naturally formed by an array of enzymes which are part of  the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and  Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm  of the cell. Recently, however, several new strains of <em>E. coli</em> have been engineered which contain an oxidizing cytoplasm  conducive to disulfide bond formation. One example of this is the SHuffle cell  line (Lobstein et al., 2012). The cell line contained mutations to key enzymes  responsible for the reducing nature of the cytoplasm, namely thioredoxin  reductase (<em>trxB</em>) and glutathione  reductase (<em>gor</em>). Further, the Shuffle  cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm.  Together, these mutations allow SHuffle to more successfully fold  disulfide-bonded proteins in the cytoplasm as compared to non-mutants. We aimed  to take advantage of disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in  cells. As shown in <strong>the figure below</strong>,  the synthesis steps may proceed as follows:</p>
<p><em>E. coli</em> naturally possesses the capacity to form disulfide bonds. In native strains,  disulfide bonds are naturally formed by an array of enzymes which are part of  the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and  Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm  of the cell. Recently, however, several new strains of <em>E. coli</em> have been engineered which contain an oxidizing cytoplasm  conducive to disulfide bond formation. One example of this is the SHuffle cell  line (Lobstein et al., 2012). The cell line contained mutations to key enzymes  responsible for the reducing nature of the cytoplasm, namely thioredoxin  reductase (<em>trxB</em>) and glutathione  reductase (<em>gor</em>). Further, the Shuffle  cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm.  Together, these mutations allow SHuffle to more successfully fold  disulfide-bonded proteins in the cytoplasm as compared to non-mutants. We aimed  to take advantage of disulfide bond forming capabilities of this strain of <em>E. coli</em> to synthesize star peptides in  cells. As shown in <strong>the figure below</strong>,  the synthesis steps may proceed as follows:</p>
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  <li> Express a short peptide containing two  cysteine residues either to the <em>E. coli</em> cytoplasm of a <em>trxB gor </em>mutant.</li>
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  <li><em>E.  coli</em> disulfide bond forming enzymes fold the peptide into a hairpin loop  structure.
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  <li>Cut the loop at a protease recognition  site engineered into the peptide. This may be done by extracting the folded  peptide from the cell and treating it with the protease in vitro. Alternatively,  the protease may be co-expressed in the cell to allow for in vivo cleavage.
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Revision as of 13:06, 17 October 2014

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Project description and results

 

Introduction and theory

Synthetic peptide chemists have long produced peptide-based materials in vitro. Star-shaped peptides are a promising type of biomaterial being explored in the field of nanomedicine (Sulistio et al., 2012). Star peptides can have several biomedical uses such as acting as drug delivery vehicles (Sulistio et al., 2011) or linkers for other biomacromolecules. Star peptides generally take the form of several linear peptide arms linked together in a central core. One way of linking these linear peptide arms together is to used covalent bonds such as disulfides. Typically, disulfide bonds are formed synthetically by taking several linear arms and treating them with an oxidant in vitro. Here, we introduce a new approach to forming star peptides by using E. coli and synthetic biology. Thus, we aimed to show how the peptides synthesis and disulfide bond forming machinery of E. coli can be used to form disulfide linked star peptide and key star peptide precursors.

 

Synthesis approach

E. coli naturally possesses the capacity to form disulfide bonds. In native strains, disulfide bonds are naturally formed by an array of enzymes which are part of the Dsb family (e.g. DsbA and DsbC) (Kadokura et al., 2003, Kadokura and Beckwith, 2009). Normally, these enzymes are found in the oxidizing periplasm of the cell. Recently, however, several new strains of E. coli have been engineered which contain an oxidizing cytoplasm conducive to disulfide bond formation. One example of this is the SHuffle cell line (Lobstein et al., 2012). The cell line contained mutations to key enzymes responsible for the reducing nature of the cytoplasm, namely thioredoxin reductase (trxB) and glutathione reductase (gor). Further, the Shuffle cell line over expresses the disulfide bond isomerase DsbC to the cytoplasm. Together, these mutations allow SHuffle to more successfully fold disulfide-bonded proteins in the cytoplasm as compared to non-mutants. We aimed to take advantage of disulfide bond forming capabilities of this strain of E. coli to synthesize star peptides in cells. As shown in the figure below, the synthesis steps may proceed as follows:

 

  1. Express a short peptide containing two cysteine residues either to the E. coli cytoplasm of a trxB gor mutant.
  2. E. coli disulfide bond forming enzymes fold the peptide into a hairpin loop structure.
  3. Cut the loop at a protease recognition site engineered into the peptide. This may be done by extracting the folded peptide from the cell and treating it with the protease in vitro. Alternatively, the protease may be co-expressed in the cell to allow for in vivo cleavage.