Part:BBa_K3114014:Design
ICARUS spacer + 6XHis tag + double terminator for purification of highly electronegative proteins
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 215
Illegal XhoI site found at 318 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1
Illegal BsaI.rc site found at 321
Design Notes
When designing circuits for the inducible expression and purification of our chlorophyll degradation pathway enzymes ((CBR: BBa_K3114024), (7-HCAR: BBa_K3114025), (SGR: BBa_K3114026), (PPH: BBa_K3114027)) we found that 7-HCAR was previously recombinantly expressed in E. coli, but catalytically inactive (Meguro et al., 2011). Through electrostatic interaction modeling of 7-HCAR, which can be read about (here), it was hypothesized that the strong electronegative binding pocket prevented 6xHis tag purification. Additionally, previous attempts to recombinantly express and purify PPH via 6xHis tag and a maltose binding protein fusion (MBP) were unsuccessful (Guyer, Salinger, Krügel, & Hörtensteiner, 2017).
To overcome these problems we sought to create a sequence that could fold between the core protein and the 6xHis tag to provide enough space that would allow for the functionality of both the protein and the purification tag.
ICARUS was designed to enable purification of large proteins with strong electrostatic potential in their binding pockets using a 6xHis-tag. The part is designed to be attached in-frame with a protein coding region at the C-terminal, which can be accomplished via Golden Gate reaction using MoClo assembly standard overhangs. The predicted structure of the universal spacer, modelled using ab initio and Rosetta comparative modelling creates a "helix-turn-helix-turn-helix" motif. SacII restriction sites are positioned at the start of the spacer and after the 6xHis-tag for removal of the 6x His-tag at the DNA level. In addition, a thrombin proteolytic site exists in the first turn of the predicted motif for 6xHis-tag removal at the protein level. The second turn in the predicted motif is filled with aspartic acid residues to repel electronegative forces, if used with a protein that has a binding pocket that is highly electronegative. However, PPH has electropositive forces within its native structure and was still shown to be purified using ICARUS fusion and was catalytically active. This part also includes a double stop codon and a double terminator (BBa_B0014). Codons were alternated to avoid tRNA depletion and were optimized based on E. coli molecular machinery.
Source
This part was synthesized.
References
Guyer, L., Salinger, K., Krügel, U., & Hörtensteiner, S. (2017). Catalytic and structural properties of pheophytinase, the phytol esterase involved in chlorophyll breakdown. Journal of Experimental Botany, 69(4), 879–889. doi: 10.1093/jxb/erx326
Meguro, M., Ito, H., Takabayashi, A., Tanaka, R., & Tanaka, A. (2011). Identification of the 7-Hydroxymethyl Chlorophyll a Reductase of the Chlorophyll Cycle in Arabidopsis. The Plant Cell, 23(9), 3442–3453. doi: 10.1105/tpc.111.089714
Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS ONE, 6(2). https://doi.org/10.1371/journal.pone.0016765