When diving into the literature for the 7-hydroxymethyl chlorophyll a reductase (7-HCAR) enzyme we discovered something shocking about its purification. 7-HCAR was previously unable to be purified through the use of a histidine tag. The inability to use histidine tag purification would introduce additional costs to enable our use of the protein. In an attempt to better understand the phenomena hindering the purification we ventured into electrostatics interaction modelling. The electrostatics modelling showed that due to charge differences between the protein and the hist tag may cause unforeseen consequences. To address these consequences we sought to develop a new spacer that would be able to withstand these forces.

Using the electrostatics interactions model we generated an “ideal” spacer that would be able to fold in such a way that it can be used for purification. Using a feedback loop oscillating between structural prediction models and wetlab review, a final spacer with the desired folding was developed. This spacer is now known as the ICARUS spacer. The ICARUS spacer has been successfully implemented for the purification of the 7-HCAR protein for which it was designed for. Along with its use with 7-HCAR, ICARUS has been successfully used on the protein pheophytinase. Along with this structural models have indicated that it maintains the desired folding on the other two proteins yet to be synthesised. With the use of the Icarus spacer our histidine tags glass wings do not melt when it gets too close to the electronegative core.

For the development of the Icarus spacer we began with an electrostatic mesh model based around the 7-HCAR protein with an unfolded spacer-His tag complex attached. This model calculates the electrostatic charge at every possible solvent interaction point, thereby generating a mesh of charges (Baker et al. 2001). From this mesh teams can qualify the long range forces acting within the protein. The electrostatic mesh was generated using the Adaptive Poission Boltzman Solver (APBS) package within Pymol 3.7.
This mesh is what alerted the team to the possible electronegativity of the protein core causing purification issues. Seen below, the blue box shows the positively charged 6xHist tag on the unfolded spacer while the red box captures the extremely electronegative core.

From this model we hypothesised that the attractive forces between the core and tag were responsible for the inhibited purification. To address this we worked with our wetlab to determine an ‘ideal’ spacer design that would fold ideally for these circumstances. Using this possibility the wetlab and drylab began to design a spacer that when folded would allow the purification of our protein. Multiple homological models were generated to slowly understand ways to induce turns and helices with desired charges.
The team decided that Alanine-Serine spacers with the first turn being a proteolytic site and the second turn being a negatively charged row of Aspartic acids. This complex then connected to the 6HIST tag and restriction site completing the entire spacer-tag system.

This system was just as expected from our homological models. The structure has also been verified through ab initio folding conducted using Rosetta on the Robetta server. The protein below is the ab initio folded Icarus spacer colour coded to resemble the diagram for the ideal spacer.