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

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.

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

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 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.

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.

Fig.4 Percentage of protein retention on PET cloth
As shown above, our fluorescent plastic-binding proteins bind to PET cloth. and stay even they are washed. In order to demonstrate in a more outstanding way, we took the movie that is shown below.

Fig.5 Plastic-binding proteins also bind to PET fiber
PET fibers were soaked in each protein solution, then washed in TBST for 5min x3. Fluorescent was observed in 460nm exciting light and imaged with 0.25sec exposure time. Magnification is 10x.

Fig.6a 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.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. The gel was CBB stained.

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.