Our project, StarScaffold, deals with star-like peptides, a recently invented class of synthetic proteins . Star-like peptides are named so because of their structure, which involves a number of linear peptide arms which branch from a central core, giving the molecule a resemblance to a pointed star. The arms are able to be ligated to various other molecules such as enzymes, therapeutic molecules, or even each other, and star-like peptides ligated to molecules in such a manner have demonstrated potential applications in areas such as drug delivery, improvement of enzyme kinetics, and novel material design.
The 2014 Melbourne iGEM team designed a novel 4-armed star-like peptide which was intended to be produced and formed largely in vivo using the bacteria E. coli and designed the peptide to contain Magainin 1 antimicrobial peptides on each of the four arms. Although the peptide was synthesised and extracted, results were ultimately inconclusive.
The 2016 iGEM team recognises the enormous potential and applications of star-like peptides, and as a result we decided to continue the work of the 2014 team. Our project, StarScaffold, features a new design with several improvements over the 2014 model, most significantly the incorporation of Split Intein motifs on the ends of each arm, as well as amino acid sequence optimisations and inclusion of new protein domains. The addition of Split Inteins importantly allows the formation of highly stable bonds in a specific and controlled manner, giving our molecule significantly improved utility.
Peptide structure and formation
The StarScaffold is transcribed as a single linear peptide, with Split Inteins in the formation shown. A cysteine residue flanks each of two Leucine Zipper regions (grey).
Through random movement, the peptide is expected to fold over itself into a hairpin loop. When this happens, the cysteine residues, with the assistance of the Leucine Zipper domains, will form a disulphide bond.
TEV protease will then cleave the protein in the middle of the loop, forming the mature star-like peptide.
The overall synthesis mechanism is very similar to the original 2014 peptide, however, the addition of Leucine Zipper domains as well as amino acid sequence optimisation alleviates a key issue of the original peptide, which was the improbability of hairpin loop and disulphide formation. We have created multiple prototypes with varying usage of Leucine Zipper domains to determine optimal formation conditions.
The formation of disulphide bonds in E. coli usually takes place within the oxidising environment of the periplasm However, mutant E. coli strains exist which are capable of facilitating disulphide bond formation in the cytoplasm, circumventing this issue. We intend to use one of these strains, Shuffle ® T7, as the ideal expression vector. Combined with in vivo expression of the TEV protease, it would potentially be possible to create a mature StarScaffold Molecule entirely in vivo.
However, given the time constraints for our project, we are currently following a more simplified synthesis pathway where the peptide is expressed in standard E. coli vectors before being extracted in its immature, linear form. Disulphide bond formation and protease cleavage will then be conducted in vitro under their respective reaction conditions.
Split intein mechanism
Inteins are a collection of natural protein splicing domains discovered in the late 20th century, which are able to undergo a self catalysed four step reaction that results in the removal of the intein domains from the host protein and the formation of a native peptide bond between their flanking sequences. As a result of this function they’ve been developed extensively for many protein splicing and immobilisation applications.
We have used this property on the arms of our star-like peptide, allowing them to be seamlessly ligated to desired molecules such as enzymes. Many different Inteins exist, and most Split Inteins from different Inteins do not cross-react, allowing us to control which specific arm a given molecule will bind to. The use of inteins also allows the star-like peptides to ligate to each other, potentially forming polymeric structures.
The StarScaffold is a foundational method of rapid, specific covalent binding and immobilisation of proteins in a fixed or unfixed orientation. The protein splicing motifs added to each end of the StarScaffold have been chosen for their ability to rapidly and selectively ligate proteins in a high strength fashion. This allows us to create an endless array new and exciting protein structures with unique and desirable properties. The two structures of immediate interest to our team are proteinaceous, permanent hydrogels and enzyme scaffold molecules.
The ability of StarScaffold molecules to ligate to each other via split intein interaction potentially allows them to form polymeric protein networks, and we intend to use this property to create a hydrogel composed of repeating subunits of StarScaffold molecules. Hydrogels are an old concept which is currently experiencing a renaissance due to new technologies and attractive applications in biomedical engineering. Hydrogels are being developed for uses such as tissue engineering in artificial organs, drug delivery, implant coatings and biomolecule purification and stabilisation.
Existing hydrogels face a number of problems such as bioincompatibility, reactive precursors, structural weakness, heat sensitivity and incorrect drug release or clearance rates. The modular nature of our system would allow the physical and biochemical properties of our hydrogel to be easily adjusted by changing star arm length or amino acid composition, or adding specialised ligands, allowing it to avoid many of the aforementioned issues, as well as potentially integrating desirable traits such as biointegration, bioerosion, cargo stabilisation, substrate selectivity, tissue adhesion and intelligent reactivity.
A trend in chemistry that has been developing over the last few decades has been to use enzymes in the production of compounds; this is usually due to overly complex substrates or insurmountable obstacles to inorganic chemical pathways. The StarScaffold can be used in these systems as a method of regulation and catalysis. Natural enzyme scaffold molecules are important in regulating the relative amounts of enzymes, increasing their stability and increasing the effective concentration of substrates for maximal rates of reaction. Perhaps the most well-known example of this is the Electron Transport Chain in cellular respiration.
Scaffold engineering is especially important where low enzyme expression levels, metabolic load or diffusion limited reactions are of concern. In similar but simpler systems rate increases of 77 fold have been seen, suggesting the StarScaffold's utility, especially for more complex chemical pathways that might be incompatible with other methods of co-localisation.
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