Difference between revisions of "Part:BBa K3185003"

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SPYCatcher

Usage and Biology

SpyCatcher is a protein that came from the CnaB2 domain of FbaB, Streptococcus pyogenes(SpyCatcher:BBa_K1159200). In a natural environment, CnaB2 domain is used for attaching to host cells. In a paper, it is partially changed and divided into two domains [1]. Nowadays, these two protein domains are known as SpyCatcher/SpyTag system because they bind irreversibly with a covalent bond.

In our experiment, we used the SpyCatcher/SpyTag system and designed only SpyCatcher part for assay(SpyCatcher:BBa_K1159200, SpyTag:BBa_K1159201). In addition, this has two tag or cleavage sites. First is 6×His-tag inserted in the N-terminus of SpyC for protein purification. Second is a TEV protease site because, in the paper, it was used for protein purification [2]. However, we didn’t use it in our experiment.

We put it between BamHI site and Ndel site on pET11-a. The expression plasmids were introduced into BL21(DE3) and expressed by T7 promoter/ T7 RNAP system. Ni-NTA agarose was used for the purification.

Sequence and Features

Purification

Fig.1 SDS-PAGE of imidazole elutes, CBB stained.

Expression

• Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37^oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.

• Protein was expressed in 0.1mM IPTG for 2hours.

Purification

1. E.coli which expressed this part were lysed with sonification.
2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).
3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.

This purification method works. As shown in Fig.1, the protein successfully purified.

Result

Fig.2 Plastic-binding protein binding to PET film
A 3µL of protein solution dropped on PET film, then left for 20min. Then the film was washed in TBST for 5min x3, then placed with Anti-His-tag-HRP conjugated for 1h. ECL substrate was added, then chemiluminescence was imaged by LAS-3000. The exposure time is 6min.

Fig.3 Isopeptide bond formation between Plastic binding proteins and Encapsulin.
3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. The gel was CBB stained.

Fig.4a Quantification of conjugated band
Conjugated bands’ intensity was quantified with ImageJ. Orange dots show averages value of three experiments. Blacklines show standard deviations. The time point 60min was deleted because it includes negative value.

Fig.4b Quantification of conjugated band Conjugated bands’ intensity was quantified with ImageJ. Orange dots show averages value of three experiments. Blacklines show standard deviations. The time point 60min was deleted because it includes negative value.

PET film assay

We tried to compare our proteins with each other by the film dot blotting.

As shown in Fig.2, the negative control protein, SpyCatcher (SPYC), did not stain PET film at all. In contrast, the plastic-binding proteins tested here strongly stained the PET film. As stains spread, we could not quantify their signals. This blot spreading might be due to the plastic-binding proteins’ fast binding rate. The proteins in excess liquid could have bound to the neighbor area of the film in the first wash step.

Although this experiment suggested our plastic-binding proteins can quickly bind to PET’s smooth surface, we could not compare binding affinity quantitatively.

Protein conjugation thorough SpyCatcher/SpyTag system

(1)Plastic-binding protein and protein capsule can be conjugated in vitro.
(2)Kinetics of SpyCatcher/SpyTag bond formation was monitored.

As we showed above, our two components: 1. Plastic-binding protein and 2. Encapsulin worked exactly as planned. Next, we conjugated these two components through SpyCatcher/SpyTag system. SpyCatcher and SpyTag form an isopeptide bond between them when they are mixed. Each plastic-binding protein is fused with SpyCatcher, and Encapsulin has SpyTags inserted on its surface (See Design).

The equal amount of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and SpyTag inserted TmEncapsulin (SpyTmEnc) solution were mixed and incubated for 16h at room temperature. Samples were taken and assessed with SDS-PAGE.

In Fig. 4a, several kinds of combinations of proteins were shown. In lane 4 and 5, SpyTmEnc is loaded with or without SpyC. Only in lane 5, which is mixed with SpyC, the upper band appeared. The molecular weight of each protein is SpyC: 15.37k, SpyTmEnc: 37.04k, so the conjugated protein should be 52.41k. We concluded that the upper band is the conjugated protein. Likewise, as shown in lane 7 and 9, SpyC-PBPs are successfully conjugated to SpyTmEnc. As the negative control, we tested TmEncapsulin without SpyTag. As expected, TmEnc and SpyC did not produce conjugated protein as shown in lane 3.

These results show we successfully conjugated several proteins to Encapsulin by SpyTag-SpyCatcher system in vitro. This means that any protein with SpyCatcher can be efficiently and easily displayed on the surface of the protein capsule.

3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. CBB-stained.

Next, we measured the time development of SpyCatcher-SpyTag bond formation. An equal amount of SpyCatcher protein (SpyC) and SpyTag inserted TmEncapsulin (SpyTmEnc) were mixed and incubated at room temperature. At different time points, 10 min, 30 min, 60 min, 180 min, 360 min, 1200 min, the reaction was stopped by adding 2x SDS sample buffer. Mixed samples were assessed with SDS-PAGE. The intensities of the conjugated bands were quantified.

As shown in Fig. 4b, conjugated bands become evident gradually (labeled with arrow). Signals were quantified and summarized in Fig. X. The reaction looks saturated after 360 minutes, even though substrates still remain a lot. This might be explained by water evaporation while incubation. Otherwise, it is possible that a bound protein prevents another protein from binding to near sites on a capsule cage. If it is the case, it might limit the number of binding proteins on a capsule.

References

1 Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., and Howarth, M. (2012).
Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.
Proc. Natl. Acad. Sci. U. S. A. <i>109.

2 Veggiani, G., Nakamura, T., Brenner, M.D., Gayet, R. V., Yan, J., Robinson, C. V., and Howarth, M. (2016).
Programmable polyproteams built using twin peptide superglues.
Proc. Natl. Acad. Sci. U. S. A.
113, 1202–1207.