When diving into the literature for the 7-hydroxymethyl chlorophyll a reductase (7-HCAR) enzyme, we discovered something shocking about its purification. Not one person has been able to His-tag purify 7-HCAR so far. The inability to use his-tag purification would introduce additional costs to our use of the protein, so we looked into ways in which we could solve this problem. In an attempt to better understand the phenomena hindering the purification of 7-HCAR, we ventured into electrostatics interaction modelling. The electrostatics modelling showed that the charge differences between the protein and the his-tag may cause unforeseen consequences. To address these consequences, we sought to develop a new spacer to sit in between the protein and the His-tag that would be able to withstand these forces.

Figure 1: vacuum electrostatic model of 7-HCAR. Blue denotes regions of electropositivity, while red denotes regions of electronegativity.

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. Along with its use with 7-HCAR, ICARUS has been successfully used on the protein pheophytinase. Structural models have indicated that ICARUS maintains its desired function on the other two proteins in the chlorophyll degradation pathway, which are yet to be synthesised.

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 to use six long, and twelve long Alanine-Serine spacers as the main portion of the protein as they are uncharged and also showed to form nice helices. To incorporate charged turns into the protein the team used a proteolytic site and a series of Aspartic acids. With the first turn being the neutral proteolytic site and the second turn being the negatively charged row of Aspartic acids. This complex then connected to a final twelve long Alanine-Serine spacer which then connected to the 6HIST tag and restriction site completing the entire spacer-tag system.

To ensure that the protein design was not biased by its repeated use of homology modelling, another method of structural prediction was used. Ab initio folding was conducted to determine the folded structure of the spacer. Folding was completed using Rosetta on the Robetta server at the University of Washington. The generated structure verified the predictions made by the team when designing the 'ideal' spacer. The protein below is the ab initio folded Icarus spacer colour coded to resemble the diagram for the ideal spacer.

After the creation of the icarus spacer had been generated it was attached to 7-HCAR, the protein for which it had been designed. With the attached Icarus spacer 7-HCAR has successfully been purified through the use of a histidine tag. With the success of Icarus the team looked to ways that Icarus could impact and benefit other iGEM teams. One possibility for the spacer to have an impact is if it were usable on other proteins. To determine if this was possible the spacer has also been attached to pheophytinase another protein previously not purified through the use of a histidine tagwithin the chlorophyll degradation pathway that also has not been previously purified through the use of a histidine tag. So far 7-HCAR(BBa_K3114025). and pheophytinase((BBa_K3114027) have been purified using the Icarus spacer. Through the use of the Icarus spacer 7-Hydroxymethyl chlorophyll-a reductase (7-HCAR) and pheophytinase are now able to be purified through the use of histidine tags.

The Icarus spacer can be found in the registry as its own part (BBa_K3114014). It can also be found in the registry connected to the parts for pheophytinase (BBa_K3114027), 7-HCAR (BBa_K3114025), Mg-Dechelatase (BBa_K3114026), and chlorophyll-b reductase (BBa_K3114024).

Future tests for the Icarus spacer involves testing on the two other proteins in the teams chlorophyll degradation pathway. Another future goal of the spacer is the characterization of its affinity with respect to other spacer systems. After characterizing and developing a vaster platform for the Icarus spacers use, our hope is that the developed part will be able to have a positive impact on iGEM projects for the future.