The aim of this project is to design and synthesise a StarScaffold, a foundational method which could be potentially used as an enzyme scaffold or a permanent hydrogel. To this end, we designed and characterised 4 parts, which are variants of the StarScaffold protein; each has its own strengths and can be chosen in different circumstances.
These two parts contain the gene sequence for our Prototype StarScaffold. These prototypes contain a single 5 repeat antiparallel leucine zipper for dimerisation as well as a set of four inteins, which were chosen to cover a range of active temperatures. This allows the progressive build-up of macromolecular structures in an effort to form proteinaceous monolayers, filaments and potentially more complicated structures. Each segment is bookended by flexible linkers to prevent steric hindrance of segment folding. This is particularly a concern for the natural split inteins found at each end of the protein as they form a folded structure before interacting. However the same is not true for the artificial split inteins which should be more amenable to internal protein segments as this is part of their native full intein structure.
This part contains the gene sequence for an alternative Double Helix StarScaffold design in which the original helix where the dimerization domain is based has been altered by lengthening the coil and changing the positions of the Asn residue used to promote antiparallel structure. The single coiled coil 'prototype' version might be preferable because of the complication to structure and disulphide bond formation caused by four helixes total in each protein. However, the dHelix part has the advantage of avoiding the increased coiled coil stability due to the lengthening of the helix in the Prototype part. It also may have superior in vivo disulphide formation thanks to the extra secondary structure around the cysteines acting to potentially exclude disulphide bond isomerases.
Sequential intein activation is not required for enzyme splicing, we have a set of four previously tested inteins we can use for enzyme co-localisation which have been shown to have a high splicing rate, yield and orthologous splicing reactions. This part contains the coding sequence for our Non-Sequential StarScaffold, which allows us to check structural changes that occur due to splicing reactions occurring in the same mixture but at alternate temperatures.
People using these parts as components of more complicated splicing systems should ensure they're using the correct intein partners for their substrate. Additionally it may be desirable to optimise linker lengths between any intein and it's extein as this can affect expression, folding and activity.