Part:BBa_K4888002

SpaC, mucus-binding protein

SpaC is a mucus-binding protein. Originating from Lactobacillus Rhamnosus GG, it has been shown to bind to the human mucus with relatively high affinity.[1]

Proof of expression in E.Coli

We expressed SpaC in E.Coli using a double control expression system, showed in BBa_K4888001.

The complete system can be seen as BBa_K4888005

As our construct contained a FLAG tag, we checked for protein expression using a western blot.

Western blot functionnement

In our experiments, the negative control consisted of transformed bacteria that had not been exposed to a specific molecule. We conducted incubation experiments using IPTG only, theophylline only, and a combination of both. Additionally, we conducted these experiments at two different temperatures, with a specific emphasis on 37 degrees Celsius, as it is crucial for our goal of expressing the protein within the human body. more information about this experiment in BBa_K4888001

Western experiment - expression of Spac using the double expression control system

Western blot analysis of SDS-boiled bacteria. The left panel displays bacteria subjected to protein induction at 18°C overnight, while the right panel shows induction at 37°C for 2 hours. 'Early induction' refers to the addition of Theophylline (Th) to the medium during the launch of the secondary culture, while 'late induction' indicates the simultaneous addition of Th and IPTG. The remaining bands were induced using either Th, IPTG, or no inducer.

Bands were detected at approximately 130 kDa, slightly above the expected 122 kDa molecular weight of SpaC, confirming successful protein expression at both 37°C and 18°C. Conversely, no signal was observed in the absence of Theophylline (Th) or Isopropyl-β-D-thiogalactopyranosid (IPTG), indicating no protein expression.

We show that the part is successfully expressed.

Trouble in Transport and Solution

To facilitate the transport of SpaC protein to the cell membrane, we initially fused it with Lpp-OmPA. Unfortunately, this approach encountered a challenge due to the size of the protein, which proved too large for effective transportation through the Lpp-OmPA system. As a result, we faced difficulties in demonstrating the correct binding of SpaC to mucus under these conditions.

In response to this issue, we conducted further investigation and discovered that SpaC comprises multiple binding domains. We hypothesized that certain domains within SpaC might be sufficient for mucus binding. To address the size limitation and enhance transport to the membrane via Lpp-OmPA, we decided to truncate the SpaC protein.[2]

The truncated proteins, including those with D2/D1 domains and those with D2 domain only, were designed and constructed. In our experiments, we successfully expressed these truncated proteins on the cell surface and observed their high binding affinity to mucus.

Shortening of SpaC, image from the paper [2]

You can find more details and information about these truncated proteins by following the provided links:

- With D2 domain only BBa_K4888003

- With D2/D1 domains BBa_K4888004

These results demonstrate the effectiveness of the truncated SpaC proteins in binding to mucus, addressing the challenges encountered with the larger protein.

References

[1] Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a humanmucus binding protein : https://pubmed.ncbi.nlm.nih.gov/19805152/

[2] Crystal structure of lactobacillar SpaC reveals an atypical five-domain pilus tip adhesin: Exposing its substrate-binding and assembly in SpaCBA pili : https://www.sciencedirect.com/science/article/pii/S1047847720301441

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1831
  Illegal PstI site found at 1871
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1534
  Illegal PstI site found at 1831
  Illegal PstI site found at 1871
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 742
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1831
  Illegal PstI site found at 1871
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1831
  Illegal PstI site found at 1871
  
• 1000
  COMPATIBLE WITH RFC[1000]