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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.

The sequence for Magainin 1 is: GIGKFLHSAGKFGKAFVGEIMKS.

When attached to a star peptide it will have the following structure:

There are several design elements to note:

• The antimicrobial peptide star will be expressed with a SUMO fusion protein. This is because without the fusion, it is likely that the peptide would be toxic to the host cell.
• The peptide includes a Factor X cutting site between the two cysteines for eventual proteolytic cleavage and formation of the star peptide.

In addition to the star peptide Magainin 1, we synthesised a gene for a linear Magainin 1 peptide as well. This is identical to the construct above, except that there is only one Magainin 1 peptide attached to the SUMO fusion.

Unstructured Peptide I (USP I) Construct

In the second approach, we designed a peptide which can be functionalised using chemical approaches. This peptide was designed to have flexible, unstructured arms and was termed the USP I. Unlike the Magainin 1 star, the arms of this peptide serve not as active peptides themselves, but as inert structural linkers.

The arms were designed with the following elements in mind:

• Lack of structure. The arms were designed with a bioinspired approach, using the FxFG motif of nucleoporins (where x is a variable amino acid residue). Such segments naturally repeat in nucleoporins  and are thought to lead to disorder/lack of stable secondary structure.  Nucleoporins are found in mammalian cells, serving as flexible brushes around nuclear pores (Ader et al., 2010).
• Water-soluble. The arms were designed with several charged amino acids to improve solubility.
• Designed to form a disulfide bond. Although it is difficult to rationally ensure that the disulfide bond will form between two cysteines in our peptide, we incorporated a beta turn between the two cysteines which may encourage the peptide to fold at the apex of the hairpin loop. This may bring the cysteines into closer proximity, providing bond formation.

The ultimate utility of this peptide lies in its ability to be functionalised with other biomacromolecules. For example, the technique of native chemical ligation can be used to join peptides, proteins, and other ligands to the arms (Dawson et al., 1994). The idea of attaching enzymes to the star peptide was explored by the University of Oxford iGEM 2014 team in a collaborative effort between our two teams[SL1].

An additional construct, a concept for an additional gene which could be proteolytically digested inside E. coli to form a star was considered. Although it [SL2] ultimately fell outside the scope of this year’s project, our work on this construct is nevertheless reported in Appendix A.