Part:BBa_K4853010
TD1-linker-V8-nisA
Lactococcus lactis nisA sequence with TD-1 construct, linker sequence, and V8 site. There is a linker sequence to help stabilize the protein. The V8 cleavage site allows the target protein to be precisely cleaved in the presence of Staphylococcus aureus.
Usage and Biology
Protein Expression
Figure 1. Coomassie-blue stained 8-18% Tris-glycine SDS-PAGE analysis of TD1-linker-V8-NisA. Lane 5 contains whole cell lysate of E. coli BL21(DE3) cells containing plasmid pRSFDuet-1-TD1-linker-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. Lane 6 contains whole cell lysate of E. coli BL21(DE3) cells containing the same plasmid with no IPTG induction. A protein band is indicated by the red asterisk at the molecular weight of 13 kDa.
E.coli BL21 (DE3) containing 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 13 kDa. While this is not the predicted molecular weight of 7.7kDa, this is consistent with previous data from Chen and Kuipers (2021)1. There is also a slight presence of a band in the non-induced sample, likely due to leaky expression of the T7 promoter.
Western Blot
Figure 2. Western blot of TD1-linker-V8-NisA. The ninth lane contains whole cell lysate of E. coli BL21(DE3) cells containing plasmid pRSFDuet-1-TD1-linker-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. The red asterisk indicates the TD1-linker-V8-NisA protein band.
To confirm the presence of TD1-linker-V8-NisA, a Western blot was conducted using a whole cell lysate of E. coli BL21(DE3) containing pRSFDuet1-TD1-linker-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 His6 tag. A band was observed at a molecular weight of 13 kDA, as indicated by the red asterisk in Figure 1. Though this band is not the predicted molecular weight of 7.7 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
1 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 136
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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