Part:BBa_K4307010

nisA-affi

Originated from Lactococcus lactis subsp. Lactis, NisA is the precursor of nisin A, a lanthionine-containing peptide antibiotic (lantibiotic) active on Gram-positive bacteria. Affibody Z_EGFR:2377 is derived from derived from staphylococcal protein A through directly evolution.^[1] We managed to design and express NisA-affibody fusion protein.
Functioned together with NisB(BBa_K4307011) and NisC(BBa_K4307012), it forms composite part NisA-affi-NisB+NisC (BBa_K4307014) to produce functional NisA-affibody peptide.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The following figure demonstrates our successful construction.

Figure 1: The construction results of NisA-affi.

Western blots was done to characterize the bio brick.

To determine the expression of NisR, we used T7 promoter and lac operator to control the overexpression of NisR by IPTG. NisR in bacterial lysate was detected by SDS-PAGE(Figure 2), indicating that NisR can be successfully expressed in E.coli.

Figure 2: Western blot results to detect NisA-affibody expression.

Conclusion

According to the western blots result, it can be found that NisA-affi can be successfully expressed in E.coli, which indicates that precursor nisin fusion protein can be produced and purified. This ensures the effectiveness of functional NisA-affibody production in E.coli.

References

[1] Friedman, M., Orlova, A., Johansson, E., Eriksson, T. L., Höidén-Guthenberg, I., Tolmachev, V., Nilsson, F. Y., & Ståhl, S. (2008). Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule. Journal of molecular biology, 376(5), 1388–1402.