Composite

Part:BBa_K3114014

Designed by: Andrew Symes, Christian Emond, Michael Wahba, Michaella Atienza   Group: iGEM19_Calgary   (2019-10-08)
Revision as of 21:47, 12 December 2019 by Christian Emond (Talk | contribs)


ICARUS spacer + 6XHis tag + double terminator for purification of highly electronegative proteins

Usage and Biology

This part can be used for fusion to the C-terminal of proteins including the ICARUS spacer and a double terminator. ICARUS can be used for the purification of large proteins that have strong electrostatic forces within their cores, using a 6xHis tag (Ni-NTA column purification). ICARUS contains flanking SacII restriction sites for removal at the DNA level and a thrombin proteolytic site for protein cleavage.

This part consists of the double terminator BBa_B0014 that has been modified to be compatible with the MoClo standard for Golden Gate assembly (Weber et al., 2011).

Figure 1. iGEM Calgary's genetic construct scheme for chlorophyll degradation pathway enzymes. Each construct consists of a T7 inducible promoter, a strong RBS, one of four chlorophyll degradation pathway enzymes, a novel spacer containing a 6xHis tag (ICARUS), and a bidirectional terminator.

iGEM Calgary engineered its chlorophyll degradation pathway enzymes to contain this ICARUS spacer to allow for successful protein purification. These parts can be found in our collection collection.

The ICARUS sequence was designed as a novel contribution to the iGEM community. ICARUS itself can be tested for its use in fusion proteins or for the purification of other proteins that have previously not been purified using a 6xHis tag.

Design

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.

400px

[Image:T--Calgary--ICARUS-Predicted-Registry.png|500px|left|][Image:T--Calgary--ICARUS-ab-initio-Registry.gif|500px|right|]

Figure 3A and 3B. Hypothesized (3A-left) and Modelled (3B-right) ICARUS Structure The left image depicts our hypothesized structure of ICARUS containing a "helix-turn-helix-turn-helix" structure. The right image is the result of ab initio structural prediction modelling.

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.

Characterization

Characterization of ICARUS can be found in its successful functionality to allow for 6xHis tag purification of our protein engineered (7-HCAR) and (PPH). Purification and characterization of these enzymes, along with additional information on ICARUS can be found (here). For more information on the in-silico characterization of ICARUS read more (here).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 215
    Illegal XhoI site found at 318
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 321

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


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