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 exposed to drastical pH change, linearized 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 protein's function is kept[1][2][3][4]. This year, we attempted to circularize 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 acids, 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. 2. Genetic construct used for the circularization of proteins using self-assembling peptide(SAP) and zip-up linker

Fig. 3. By using glutathione-S-transferase, 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 at the N and C terminal are brought closer to each other 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-terminus and the C-terminus varies depending on the proteins, we need to prepare linkers with appropriate length. 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 shown in Fig. 4 is obtained.

1. Designing constructs

We designed one construct for circularization of a protein and 3 constructs as negative controls. The complete construct is shown in Fig. 5. We chose GFP as the protein part for assay of this biodevice. Two of the negative control constructs, ΔSAR and ΔZip, lack SAR or zip-up linker, and ΔSAR・Zip has only GFP coding sequence with His-tag, as shown in Fig. 6. We designed ΔSAR and ΔZip construct to assess the effect of SAR or zip-up linker. If both parts definitely contribute to efficiency of circularization, the products from these two constructs show less thermostability compared to the products from the complete construct. ΔSAR・Zip construct has only GFP and His-tag sequence so they wouldn’t show improved thermostability. As you can see in the Fig. 5, the complete construct has some restriction enzyme recognition sites, so you can make the three other constructs by digestion with appropriate restriction enzymes.

Fig. 5. The complete construct for protein circularization

Fig. 6. The constitutions for negative controls

2. Making circuits

We ordered DNA sequence of the complete construct from IDT. As shown in Fig. 7, we planned to ligate firstly the complete construct with pSB1C3 vector after PCR and subcloning, and then make three other constructs by digestion with appropriate restriction enzymes.

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

3. IPTG induction, purification and circularization

Add IPTG into cultured bacteria in liquid medium. After confirming expression of GFP, the homogenate which includes GFP will be obtained by causing cell lysis with freeze-thaw. As the products from our 4 DNA constructs have His-tag, you can purify them with Ni-affinity chromatography. After purification of protein, incubate the protein solution under suitable physiochemical conditions to induce self-assembling of the distal SAR of a protein, and then treat with glutathione-S-transferase to form disulfide bonds between cysteine residues in the zip-up linkers.

Fig. 8. Purification and circularization of protein

4. Assay

First, you need to calculate the concentration of each sample and make it even among 4 samples; product from the complete construct and three negative control constructs. Then treat the protein solution with heat shock at desired temperature of around 50~70°C for example, for 1 to 5 seconds in PCR cycler. Besides, you need one control sample to treat at 37°C for each construct. After heat shock treatment, cool down the samples at 37°C and compare the fluorescence intensity of each sample.

[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. & Pluckthun, A. Circular beta-lactamase: stability enhancement by cyclizing the backbone. FEBS Lett. 459, 166-172 (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, 16548-16554 (2001).