Part:BBa_K4853011
Truncated Leader
This sequence encodes the bare minimum motif recognized by the post-translational modifiers NisB and NisC within the leader peptide of nisin A. Dehydration and cyclization reactions begin 13 amino acids downstream of the motif.
Usage and Biology
To test the expression and functionality of our truncated leader sequence, we placed the sequence in the same plasmid as nisA. The full plasmid contains the truncated leader sequence, TD-1 sequence along with a combination of a WELQ site, and the nisA sequence. Through SDS-PAGE analysis, we confirmed expression of the open reading frame containing the truncated leader, indicated by the red asterisks in Figure 1.
Protein Expression
Figure 1. Coomassie blue-stained 8-18% Tris-glycine SDS-Page analysis of TD-1-NisA constructs. Lane 1 contains whole cell lysate of E. coli BL21(DE3) pRSFDuet-1-TL-TD1-V8-nisA incubated with 0.4 mM IPTG at 37°C for 4 hours. Lane 2 contains the whole cell lysate of E. coli BL21(DE3) pRSFDuet-1-TL-TD1-V8-nisA with no IPTG induction. Protein bands are indicated by the red asterisks in lane 1 at an approximately molecular weight of 11 kDA respectively.
To confirm the functionality of the truncated leader, 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 (Fig.2). Our construct has a His6 tag as well. When NisB and NisC are co-expressed with TL-TD1-V8-NisA, NisA undergoes some modifications evidenced by the molecular weight of the band changing from ~11 to ~100 kDa. This change is the same for NisA alone when co-expressed with NisB and NisC. Because NisB and NisC modify NisA in the same way whether using the original leader peptide or our truncated leader sequence, NisB and NisC most likely recognize the truncated leader sequence.
Western Blot
Figure 2. Western blot of TL-TD-1-NisA constructs. The first 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 second lane contains whole cell lysate of E. coli BL21(DE3) cells containing plasmid pRSFDuet-1-TL-TD1-V8-nisA incubated without any IPTG. The presence of the bands of interest is indicated by a red asterisk.
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 Plat, A., Kluskens, L., Kuipers, A., Rink, R., Moll, G. (2011). Requirements of the Engineered Leader Peptide of Nisin for Inducing Modification, Export, and Cleavage. Appl Environ Microbiol, 77(2), e604-611. https://doi.org/10.1128/aem.01503-10
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |