Team:Melbourne/Project

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<h1>Project description and  results</h1>
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<h1 >Project</h1>
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<h2>Project description and  results</strong></h2>
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<h3><strong>Introduction and theory</strong></h3>
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<h2>Introduction and theory</h2>
<p>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 <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and  disulfide bond forming machinery of <em>E.  coli</em> can be used to form disulfide linked star peptide and key star peptide  precursors.</p>
<p>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 <em>E. coli</em> and synthetic biology. Thus, we aimed to show how the peptides synthesis and  disulfide bond forming machinery of <em>E.  coli</em> can be used to form disulfide linked star peptide and key star peptide  precursors.</p>
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<h2>Synthesis approach</h2>
<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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<p>This synthesis approach has several benefits over purely  in vitro approaches. Firstly, the exact peptide sequence can be precisely  programmed into <em>E. coli</em> using  recombinant DNA synthesis. Secondly, by performing the disulfide bond formation  in cells and optionally the proteolytic cleavage, several synthesis steps which  would need to be performed in vitro are eliminated. From a scale up  perspective, this would eliminate entire unit operations which would otherwise  be required to produce this product. Given these benefits, in the current study,  we aimed to express a star-peptide precursor to the cytoplasm of Shuffle cells  to later be extracted and externally digested with the star-forming protease.  In order to achieve this, we first designed several star-peptides which might  be amenable to this synthetic strategy. These peptides are described below.</p>
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<h2>Rationally designed peptides</h2>
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<p>There are two approaches to functionalising star  peptides. In the first, the identity of the arms can be chosen to be bioactive  peptide molecules. This is the simplest approach to producing a  biologically-relevant star. In the second, the arms can be functionalised by  ligating molecules to them at any point. We used both of these approaches  designed to separate peptides.</p>
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<h3>Magainin 1 star and linear peptides</h3>
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<p>Our first strategy was to make a star peptide using  antimicrobial peptides as building blocks. Antimicrobial peptides (AMPs) are  small, approximately 50 residue peptides secreted by some bacteria and  mammalian cells which selectively kill microbial cells. It is thought that AMPs  work by forming pores in the membrane of prokaryotic cells (Brogden, 2005).  AMPs have been recombinantly expressed in a number of organisms, including <em>E. coli</em> (for a review, see Li, 2011) and <em>B. Subtilis</em> (Chen et al., 2009, Yu et  al., 2013).<br>
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  Our concept was to design a star peptide with  antimicrobial peptide arms. Wiradharma et al. (2012) first showed that placing  linear antimicrobial peptides in a star configuration could lead to enhanced  antimicrobial activity and decreased hemolytic activity. Although it is unclear  why this is the case, it may be due to the ability of neighboring antimicrobial  peptide arms to interact with each other to synergistically rupture the  membrane.<br>
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While Wiradharma used a synthetic peptide sequence, we  designed a peptide using the naturally occurring AMP, Magainin 1 (Zasloff,  1987). Magainin 1 peptides will be placed to the ends of each star arm.</p>
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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.

 

 

This synthesis approach has several benefits over purely in vitro approaches. Firstly, the exact peptide sequence can be precisely programmed into E. coli using recombinant DNA synthesis. Secondly, by performing the disulfide bond formation in cells and optionally the proteolytic cleavage, several synthesis steps which would need to be performed in vitro are eliminated. From a scale up perspective, this would eliminate entire unit operations which would otherwise be required to produce this product. Given these benefits, in the current study, we aimed to express a star-peptide precursor to the cytoplasm of Shuffle cells to later be extracted and externally digested with the star-forming protease. In order to achieve this, we first designed several star-peptides which might be amenable to this synthetic strategy. These peptides are described below.

 

Rationally designed peptides

There are two approaches to functionalising star peptides. In the first, the identity of the arms can be chosen to be bioactive peptide molecules. This is the simplest approach to producing a biologically-relevant star. In the second, the arms can be functionalised by ligating molecules to them at any point. We used both of these approaches designed to separate peptides.

Magainin 1 star and linear peptides

Our first strategy was to make a star peptide using antimicrobial peptides as building blocks. Antimicrobial peptides (AMPs) are small, approximately 50 residue peptides secreted by some bacteria and mammalian cells which selectively kill microbial cells. It is thought that AMPs work by forming pores in the membrane of prokaryotic cells (Brogden, 2005). AMPs have been recombinantly expressed in a number of organisms, including E. coli (for a review, see Li, 2011) and B. Subtilis (Chen et al., 2009, Yu et al., 2013).
Our concept was to design a star peptide with antimicrobial peptide arms. Wiradharma et al. (2012) first showed that placing linear antimicrobial peptides in a star configuration could lead to enhanced antimicrobial activity and decreased hemolytic activity. Although it is unclear why this is the case, it may be due to the ability of neighboring antimicrobial peptide arms to interact with each other to synergistically rupture the membrane.
While Wiradharma used a synthetic peptide sequence, we designed a peptide using the naturally occurring AMP, Magainin 1 (Zasloff, 1987). Magainin 1 peptides will be placed to the ends of each star arm.