Part:BBa_K4853012

truncated leader + TD1+ V8 + nisA

Lactococcus lactis nisA sequence with WELQ site, TD1 construct, truncated leader sequence and V8 site. The WELQ protease cleavage site allows for cleavage of an N-terminal epitope tag that may be added. The V8 cleavage site allows the target protein to be precisely cleaved in the presence of Staphylococcus aureus.

Protein Expression

Figure 1. Coomassie-blue stained 8-18% Tris-glycine SDS-PAGE analysis of TL-TD1-V8-NisA. Lane 1 contains whole cell lysate of E. coli BL21(DE3) cells containing plasmid pRSFDuet-1-TL-TD1-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. Lane 2 contains whole cell lysate of E. coli BL21(DE3) cells containing the same plasmid construct, with no IPTG induction. Note the presence of a band at 12 kDa indicated by the red asterisk.

E.coli BL21 (DE3) containing TL-TD1-linker-V8-nisA was induced with 0.4 mM IPTG. As seen in figure 1, the protein band was observed at a molecular weight of 12 kDa. While this is not the predicted molecular weight of 6.7 kDa, this is consistent with previous data from Chen and Kuipers (2021). There is also a slight presence of a band in the non-induced sample, likely due to leaky expression of the T7 promoter.

Figure 2.Western blot of TL-TD1-V8-NisA. The fifth lane contains whole cell lysate of E. coli BL21(DE3) cells containing plasmid pRSFDuet-1-TL-TD1-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. The red asterisk indicates the TL-TD1-V8-NisA protein band.

To confirm the presence of TL-TD1-V8-NisA, a Western blot was conducted using a whole cell lysate of E. coli BL21(DE3) containing pRSFDuet1-TL-TD1-V8-nisA. The positive control used was a Posi-Tag, which has several proteins with different epitopes, including His₆. Our construct was made with an N-terminus His₆tag. A band was observed at a molecular weight of 12 kDA, as indicated by the red asterisk in Figure 1. Though this band is not the predicted molecular weight of 6.7 kDa, the band presented is consistent with previous data from Chen and Kuipers (2021). The presence of the His₆ tag at the same molecular weight as seen on the SDS-PAGE gel allows us to conclude that our E. coli BL21(DE3) cells were expressing the desired protein.

References

Chen, J., & Kuipers, O. P. (2021). Isolation and Analysis of the Nisin Biosynthesis Complex NisBTC: Further Insights into Their Cooperative Action. mBio, 12(5), e02585-21. https://doi.org/10.1128/mBio.02585-21

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 167
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]