Part:BBa_K3185006

SPYCatcher -> sfGFP -> LCI KR-2

Usage and Biology

LCI is a protein from Bacillus subtili. The paper shows that it can bind to polypropylene(PP) [1]. Another paper shows the improved variant, LCI-KR2(Y29R and G35R; variant KR-2) [2]. Its affinity is 5.4±0.5 times stronger than natural LCI.

We used LCI-KR2 for binding protein to PP. We inserted superfolder GFP (sfGFP) which folding interval is shortened by improving natural GFP on the N-terminus of LCI (BBa_I746916). By doing so we wanted to do the binding assay with fluorescence. Moreover, we put SpyCatcher(BBa_K1159200)[ on N-terminus of sfGFP because we used SpyCatcher/SpyTag system to bind it to other parts.

This part has four tags. First is 6×His-tag inserted on the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between sfGFP and Spy-Catcher to detect it by using the antibody. The third is a TEV protease site and we put it into two regions because it was used for protein purification in the paper[3].

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1174
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 460
  

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 with serial dilution, then left for 20min. Then, the film was washed in TBST for 5min x3, then placed with Anti-His-tag-HRP conjugated for 1h. After washing, ECL substrate was added, then chemiluminescence was imaged by LAS-3000. The exposure time is 6min.

Fig.3a Cloth dot blot by fluorescent plastic-binding protein before washing.
The dilution collection of each protein was dropped on PET cloth, then left for 20min. The protein fluorescent was imaged by LAS-3000. The exposure time is 10sec. Saturated signals were painted red.

Fig.3b Cloth dot blot by fluorescent plastic-binding protein after washing.
The dilution collection of each protein was dropped on PET cloth, then left for 20min. The cloth was washed in TBST for 5min x3, then protein fluorescent was imaged by LAS-3000. The exposure time is 10sec. Saturated signals were painted red.

Fig.4 Percentage of protein retention on PET cloth
The whole signal of 37.5ng dot was used for this quantification, percentages of protein retention were calculated by comparing intensity before and after washing.

Fig.5 Plastic-binding proteins also bind to PET fiber
PET fibers were soaked in each protein solution, then washed in TBST for 5 min x 3 times. Fluorescence was observed in 460 nm exciting light and imaged with 250 ms exposure time. Magnification is 10x.

Fig.6a SDS-PAGE gel for quantification of amounts of proteins bind to PET fiber
20 cm of PET fibers were soaked in protein solutions, then washed in TBST for 5 min three times. Washed fibers were soaked in 50µL of 2x SDS sample buffer. Bound proteins were eluted with boiling. SDS-PAGE for 40min in 200V. CBB-stained.

Fig.6b BaCBM2 bind 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.7 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. 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 cloth assay

Next, we quantified how much protein binds to PET fiber with fluorescence. LCI proteins can be seen with fluorescent signals because it is fused with sfGFP.

We bought a white PET T-shirt and cut it into pieces. The lysate of E. coli which expresses protein is used for this experiment. The concentrations of fluorescent proteins were measured by SDS-PAGE and CBB-staining, and an equal amount of proteins were used for each assay. Diluted proteins were spotted on a piece of PET cloth. The cloth was incubated for 20 min at room temperature, then washed by TBST for 5 min 3 times. Finally, fluorescent proteins were photographed.
Fig.3a shows the cloth before wash, and 3b shows the same cloth after wash. As shown in figures, sfGFP was completely washed out. In sharp contrast, LCI strongly stuck to PET cloth. The intensity of dot was quantified with ImageJ.
As shown in Fig.4, about 65% of LCI KR-2 were still observed on PET cloth after wash, showing these proteins are really strong PET cloth binders.

Based on the data shown above, we concluded that our fluorescent plastic-binding proteins highly stably bind to PET cloth. In order to demonstrate this conclusion clearly, we drew a picture by two different GFP inks; sfGFP alone and GFP-LCI KR-2. When washed by TBST, sfGFP is washed out, while the stable PET binder GFP-LCI KR-2 remains. See this movie! Our mascot "KonKon" will appear! File:Konkon.mp4

PET fiber assay

We showed two fluorescent plastic-binding proteins bind to PET cloth very tightly. Next, 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 mins 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.

Clearly, when LCI KR-2 were fused to sfGFP, the PET fiber was brightly stained by fluorescence. Water control did not show any fluorescence, indicationg that no autofluorescence was observed with these fibers. sfGFP control also showed no signals, meaning that sfGFP binding was mediated by the plastic-binding domain in the sfGFP fusion proteins.

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.6a and 6b, BaCBM2 binds the most to PET fiber. According to the references, BaCBM2 and CenA is a polyethylene terephthalate (PET)-binding protein, LCI KR-2 is a polypropylene (PP) binding protein, and TA2 is a polyurethane (PU) binding protein. Therefore, this result is consistent with the reported observation. It is of interest that LCI KR-2 (PP binding) and TA2 (PU binding) proteins also showed a moderate but measurable amount of PET-binding.

Protein conjugation thorough SpyCatcher/SpyTag system

Next, we conjugated SpyC->LCI KR-2 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. 7, 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 Rübsam, K., Stomps, B., Böker, A., Jakob, F., and Schwaneberg, U. (2017).
Anchor peptides: A green and versatile method for polypropylene functionalization.
Polymer (Guildf). 116, 124–132.

2 Rübsam, K., Davari, M.D., Jakob, F., and Schwaneberg, U. (2018).
KnowVolution of the polymer-binding peptide LCI for improved polypropylene binding.
Polymers (Basel). 10, 1–12.

3 Rübsam, K., Weber, L., Jakob, F., and Schwaneberg, U. (2018).
Directed evolution of polypropylene and polystyrene binding peptides.
Biotechnol. Bioeng. 115, 321–330.