Part:BBa_K4853002
WELQ + V8 cleavage site + nisA
Lactococcus lactis nisA sequence with a WELQ site and V8 cleavage 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.
Usage and Biology
Figure 1. Western blot of V8-NisA. The first lane contains whole cell lysate of E. coli BL21(DE3) cells containing plasmid pRSFDuet-1-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. The red asterisk indicates the V8-NisA protein band.
To confirm the presence of the nisA protein, a Western blot was conducted using a whole cell extract collected directly from E. coli BL21(DE3) containing pRSFDuet1-V8-nisA. The positive control used was a Posi-Tag, which has several proteins with different epitopes, including one for His6. Our construct was made with a His6 tag. With respect to the V8-nisA construct, a band was observed at a molecular weight of 12 kDA, as indicated by the red asterisk in Figure 1. While this is not the predicted molecular weight of 5.81 kDA, this is consistent with previous data from Chen and Kuipers (2021).
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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 91
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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