Team:HokkaidoU Japan/Circularization

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Team:HokkaidoU Japan

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• Top
• Project
  • Overview
    • Project Description
    • Background
    • Proof of concept
    • Modeling
    • Achievements
  • MULTIMERIZATION
    • Overview
    • Methods
    • Results
    • Conclusion
  • FUTURE WORK
    • Circularization
    • Kill Switch
    • Transport
    • Aggregation
  • Extra Materials
    • Parts
    • Notebook
    • Protocols
    • Safety
• Human practices
• Collaborations
• Team
  • Attribution
  • Member

Although enzymes are essential for the development of science, they can only be used under certain conditions due to its nature that it inactivates depending on the temperature and pH. Ways to gain enzymes that are stable against such conditions include investigation of microorganisms that survive under extreme environments, improvement of the activation using amino acid substitution, compartmentalization of the enzymes, and circularization of polypeptides. When enzymes are heated or the pH changed, linear-type enzymes are denatured and deactivated. On the other hand, with circularized enzymes, it is believed that since the ends of the polypeptide are joined and protected, the tertiary structure is less likely to be broken and the activation kept[1][2][3][4]. This year, we attempted in the circularization of proteins using self-assembling peptide(SAP) and zip-up linker.
The SAPs we used are RADA-16-I and P₁₁-4. These are both artificially created amphiphilic SAPs, consisting of amino acid sequence RADARADARADARADA and QQRFEWEFEQQ respectively. They self-assemble under suitable physiochemical conditions due to the polar amino acids and hydrophobic interaction and form β-sheet (Fig. 1).

Fig. 1. RADA16-I and P₁₁-4 self-assemble under suitable physiochemical conditions due to the polar amino acids and hydrophobic interaction and form β-sheet.

By creating a construction as shown in Fig. 2, the self-assembling region(SAR) and the region containing the SAP interact, thus bringing closer the zip-up linkers on the N-terminal and the C-terminal (Fig. 3).

Fig. 2. Genetic construct used for the circularization of proteins using self-assembling peptide(SAP) and zip-up linker

Fig. 3. By using glutathione-S-transeferase, circularization of polypeptides are obtained.

Fig. 4. A structure of circularization of polypeptides

The zip-up linker plays a vital role in the creation of the covalent bond essential for the circularization of proteins. This consists of amino acid sequence of CWEGGGCGGGCGGGCSALCGGGCGGGCGGG, and is composed of repetition of 3 glycine and 1 cysteine residues. We are hoping that the zip-up linker on the N and C terminal are brought closer by the SAR, and that the cysteine residues form disulfide bonds from the SAR as if to zip up the ends.
Since the distance between the N-terminal and the C-terminal varies depending on the protein, it is essential that a linker of an appropriate length is chosen. This would usually mean that it is necessary to change the linker depending on the protein that is to be circularized. However, our zip-up linker has enough GGGC sequence so that only the flexible part of the linker form disulfide bonds, thus preventing deformation of the tertiary structure. This means that a suitable length of the linker will be used to suit the structure of each protein, enabling this zip-up linker to be applied to various proteins regardless of its structure. Eventually, a structure as indicated in Fig. 4 is obtained.

1. Designing constructions

We designed one constructction for circularization of a protein and 7 constructions for negative control. Circularization construct has the same structure as the construct shown in Fig. 2 We chose GFP as the protein part for assay of this biodevice. The negative control construct is shown in Fig. 5-Fig. 11. Each construct lacks one part of the complete one. つづく

Fig. 5. The complete construction for protein circularization

Fig. 6. The constitutions for negative control

2.

3. IPTG induction

4. Purification

5. Circularization

6. Assay

Fig. 7. Method for making complete circuit and negative control circuits

Fig. 8. Purification and circularization of protein

[1] Scott, C. P., Abel-Santos, E., Wall, M., Wahnon, D. C. & Benkovic, S. J. Production of cyclic peptides and proteins in vivo. Proc. Natl. Acad. Sci. 96, 13638–13643 (1999).
[2] Iwai, H. & Plückthun, A. Circular beta-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett. 459, 166–72 (1999).
[3] Flory, J. & Yol, S. Theory of Elastic Mechanisms in Fibrous Proteins. 715, 5222–5235 (1956).
[4] Iwai, H., Lingel, a & Pluckthun, a. Cyclic green fluorescent protein produced in vivo using an artificially split PI-PfuI intein from Pyrococcus furiosus. J. Biol. Chem. 276, 16548–54 (2001).