Difference between revisions of "Part:BBa K3185004"

Latest revision as of 02:07, 22 October 2019

SPYCatcher -> BaCBM2

Usage and Biology

BaCBM2 is a Carbohydrate-Binding Module (CBM) from Bacillus anthracis. CBM often found in Carbohydrate related enzymes. It can bind to not only highly crystallized cellulose but also PET because it has a binding site formed by aromatic amino acids [1]. In this paper, they research binding affinity of some kinds of CBM and PET. As a result, it is found that BaCBM2 has the most strong binding affinity to PET [2].

We used BaCBM2 as PET binding domain. We put SpyCatcher on N-terminus of BaCBM2 because we used SpyCatcher/SpyTag system to bind it to other parts(SpyCatcher:BBa_K1159200, SpyTag:BBa_K1159201). Also, this has three tag and cleavage sites. First is 6×-His tag inserted in the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between SpyC and CBM to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification [3]. 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

Fig1.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.3a SDS-PAGE gel for quantification of amounts of proteins bind to PET fiber 20cm of PET fibers were soaked in protein solutions, then washed in TBST for 5min three times. Washed fibers were soaked in 50µL of 2x SDS sample buffer. Bounded proteins were eluted with boiling. SDS-PAGE for 40min in 200V. CBB stained.

Fig.3b BaCBM2 binds most to PET fiber
SDS-PAGE’s gel band intensity quantified with ImageJ. The y-axis shows amounts of protein which bind to 20cm PET fiber.

Fig. 4 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.

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.

PET fiber assay

We demonstrated proteins’ binding in a more realistic target: PET fiber. In cloth, fibers are close to each other, so they might create a hydrophobic environment between them. In the fiber form, they are surrounded by water, so plastic-binding proteins might behave in a different way.

In this experiment, PET fibers were soaked in water or protein solutions, then washed in TBST for 5 min three times. The concentrations of protein solutions were 2000 ng/µL. sfGFP and other sfGFP-fused protein’s fluorescence were observed by a fluorescence microscope in 460 nm exciting light.

We next compared with other plastic-binding proteins quantitatively. The equal length of PET fibers ware soaked in protein solutions and proteins bound were visualized in SDS-PAGE and CBB stain.

As shown in Fig.3a and 3b, BaCBM2 binds the most to PET fiber. According to the references, BaCBM2 is a polyethylene terephthalate (PET)-binding protein. Therefore, this result is consistent with the reported observation.

Protein conjugation thorough SpyCatcher/SpyTag system

We conjugated SpyC->BaCBM2 with SpyTag inserted TmEncapsulin (BBa_K3185000) through SpyCatcher/SpyTag system. SpyCatcher and SpyTag form an isopeptide bond between them when they are mixed. SpyTag inserted TmEncapsulin has SpyTags inserted on its surface.

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. 4, 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. 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.

References

1 Boraston, A.B., Bolam, D.N., Gilbert, H.J., and Davies, G.J. (2004).
Carbohydrate-binding modules: Fine-tuning polysaccharide recognition.
Biochem. J. 382, 769–781.

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.

3 Weber, J., Petrović, D., Strodel, B., Smits, S.H.J., Kolkenbrock, S., Leggewie, C., and Jaeger, K.E. (2019).
Interaction of carbohydrate-binding modules with poly(ethylene terephthalate).
Appl. Microbiol. Biotechnol. 103, 4801–4812.