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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.

Expression system

In order to successfully express our constructs, we designed our protein expression vectors to include a fusion protein. The fusion protein was necessary for two reasons. First, some of our constructs are very small (e.g. the non-star Magainin 1), and expression levels of very small peptides can be difficult without a fusion partner. Second, two of our constructs code for antimicrobial peptides. Without a fusion partner, it is likely that these genes would be toxic to their hosts upon induction.

To find a suitable expression system, we looked towards the Registry of Standard Parts. We used the SUMO protein expression system designed by TU Delft 2014 (for example, seehttp://parts.igem.org/wiki/index.php?title=Part:BBa_K1022101). This system essentially consists of a N-terminal HIS-tag followed by the SUMO protein (also known as UlpI).

The strategy of expressing toxic AMPs using SUMO has been successfully reported in the literature (Bommarius et al., 2010). We surmise that the SUMO protein could inhibit the antimicrobial activity of single, linear peptides, that it may also inhibit the activity of our star antimicrobial peptide.

SUMO as a fusion protein also has the benefit of leaving no residues at the C-terminal end of the cleavage site. This means that upon cleavage with the SUMO protease, the native protein can be recovered. In our case, this means that one of the arms of the star can be designed without the need to take into account the addition of any amino acid residues left behind by the protease.
We used the SUMO peptide sequence reported by TU Delft. However, our construct contained the following unique features:

1. Standardised the biobrick by substituting the T7 promoter and RBS (the origins of which are both not specified in the Delft documentation) with the standard T7 promoter and RBS BioBrickBBa_K525998. In addition to supporting the principle of standardization, using the well-characterized promoter BioBrick should help assure expression levels.
   
2. Biobrick BBa_K1022101 lacks a terminator sequence (this was presumably because the part was meant to be integrated into a larger genetic construct with a terminator). A terminator from the registry of standard parts was added (specifically, the wild type terminator from T7 bacteriophage,BBa_K731721).
   
3. The original biobrick BBa_K1022101 codes for three amino acid residues before the his-tag (ASM), which appeared to be redundant. Correspondence with the 2013 TU Delft team suggested that these residues were unnecessary and appear to be cleaved within the cell as part of the cells post-translational modifications. However, their presence complicates the addition of additional tags at the N-terminus of the protein (e.g. periplasmic export tags), and therefore they were not included.
   
4. The SUMO sequence was codon optimised for E. coli during synthesis. As the Delft documentation did not specify whether the gene was codon optimal, codon optomisation was undertaken to potentially improve expression levels. The linear Magainin 1 construct, however, was not codon optimised in the SUMO region in order to provide a control condition.

To summarise, the protein expression devices used in our project to the following form:

The protein coding region consisted of a 6x HIS tag followed by the SUMO protein and the relevant star or linear peptide.

Plasmid preparation: Cloning and acquisition of the genetic constructs

Magainin 1 Star Peptide

This construct was synthesised in the standard shipping plasmid from Life Technologies- plasmid pMK. We had originally planned to express our protein to the periplasm of E. coli, and therefore had included in the synthesis a periplasmic export tag, the TorTss signally sequence (Steiner et al., 2006).

Cytoplasmic and periplasmic expression may both allow for disulfide bond formation in the cell. We decided, however, to focus on cytoplasmic expression. There are distinct advantages to cytoplasmic expression (e.g. the absence of several periplasmic proteases and potentially higher expression levels (Baneyx, 1999)).

Another reason the cytoplasm was chosen was to allow us to test the effects of an oxidising versus reducing intracellular environment on disulfide bond formation. We planned to express the construct in both SHuffle T7 cells (oxidising cytoplasm) and BL21(DE3) (reducing cytoplasm) to probe whether there was a difference in disulfide bond formation. Therefore, we needed to remove the periplasmic export tag from the gene.

To do this, we use the following cloning strategy (noting that the top row represents the gene initially synthesised in plasmid pMK):

The gene was inserted into plasmid pSB1C3 containing the T7 promoter and ribosome binding site, BBa_K525998. It was inserted after the promoter and before the BioBrick suffix. This was accomplished by digesting the destination vector with SpeI and PstI. At the same time, PCR was used to amplify the segment of the gene containing the SUMO fusion and the Magainin 1 Star peptide, adding XbaI and keeping the PstI site existing in the gene.

Digestion of the PCR product then allowed for ligation of the insert and destination vector using the PstI sites and XbaI and SpeI (XbaI and SpeI have compatible sticky ends). Note that after the ligation, there will be a scar in the gene where the XbaI and the SpeI sites were ligated.

The ligation appeared to be successful. The ligation mixture was transformed to DH5α competent cells and plated onto chloramphenicol-containing agar plates.

Plasmid from 5 colonies (C1, C2, C3, C4, and C5) was cultured, extracted, and then digested with EcoRI and PstI.

As evident in the figure below, at least 4 of the colonies appeared to contain the insert. The empty, linearised pSB1C3 backbone ran at approximately the correct size (2070 bps), as did the insert (740 bps).

Notes: MW marker is the 100 bp Ladder from Axygen. * indicates the colony picked for sequencing and eventual transformation to the expression cell lines.

The DNA from Colony C2 was confirmed using Sanger sequencing at the Australian Genome Research Facility. This DNA appears in the registry of standard parts as BioBrickBBa_K1394000.

For the USPI peptide and the linear Magainin 1 peptide, the genes were synthesised by GenScript and delivered in pSB1C3. The expression vectors had identical gene regulatory elements to that used for the Magainin 1 Star peptide. They only differed in the codon optimisation used, and they also lacked the assembly scar described above.