Part:BBa_K5136221
I0500-B0034-FhuD-GGG linker-T7 lysozyme 119V-SsrA-B0015
Biology
FhuD
In Escherichia coli, protein translocation guided by signal peptides primarily employs two distinct mechanisms: the Sec- pathway and the Tat- pathway. Notably, some proteins are capable of utilizing both pathways for their translocation (1) . The FhuD signal peptide, acting as an intrinsic dual Sec-Tat pathway (2), is frequently employed in biotechnological applications to direct the secretion of proteins to the extracellular space or the cell membrane. This characteristic makes the FhuD signal peptide an ideal choice for constructing secretion expression vectors, particularly in applications aimed at enhancing the yield of target proteins.
GGG Linker
[(G4S)n] is commonly used in protein engineering because of its flexibility and resistance to proteases. Therefore, we selected (GGGGS)3 flexible linker (3) as a short peptide to connect FhuD and T7 lysozyme 119V in our autolytic system.
T7 Lysozyme 119V
T7 lysozyme is a small molecular weight protein in bacteriophage T7, primarily functioning to degrade the cell wall of host bacteria during phage infection, facilitating the injection of phage DNA or the release of newly formed phage particles. In molecular biology research, it is widely used for the efficient lysis of Escherichia coli cells (3, 4). Moreover, it has been reported that higher levels of lysozyme provided by plasmids pLysE or pLysH can reduce the full induction activity of T7 RNA polymerase, allowing induced cells to continue growing indefinitely while producing non-toxic target proteins (4). This feature not only highlights the excellence of T7 lysozyme in promoting cell lysis but also makes it extremely useful in preparing cell extracts for protein purification.
Notably, T7 lysozyme 119V was selected from the UniProt database (6), and it differs from the T7 lysozyme 119G sequence found in pLysS (7), with a variation at the 119th amino acid position.
SsrA
The SsrA is a small peptide tag used to mark proteins for protein degradation. When fused with the target protein, SsrA could guide it to specific proteases, such as the ClpXP and ClpAP complexes, for degradation (7).
Usage and Design
In our design, we aim to induce cell autolysis to release enzymes into the supernatant, simplifying the complex protein purification process. By utilizing the dual-pathway signal peptide FhuD, we direct T7 lysozyme to the peptidoglycan layer, enhancing cell lysis. Additionally, the SsrA tag is fused to the C-terminus of T7 lysozyme to ensure the degradation of any leaked T7 lysozyme, minimizing system cytotoxicity and ensuring the proper accumulation of the target enzyme in the correct location (8).
This composite part we constructed aims to express the FhuD-T7 lysozyme-SsrA mediated autolytic system (FLSA), which includes T7 lysozyme 119V, under the control of an L-arabinose inducible promoter. To validate the efficiency of the FLSA system, we used sfGFP as a reporter.
Characterization
Agarose Gel Electrophoresis (AGE)
The composite part (BBa_K5136221) constructed was introduced into the backbone plasmid (pSB1C3) through standard assembly and transformed into E. coli DH10β. The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (2332 bp) can be observed at the position around 3000 bp. (Figure 2).
sfGFP Release Efficiency Determination
After co-transforming I0500-B0034-FhuD-TSS linker-T7 lysozyme 119G-SsrA-B0015_pSB1C3 and sfGFP_pET-28a(+) into E. coli BL21(DE3), the cultures were grown overnight in LB medium containing corresponding antibiotics. The cultures were diluted and grown to OD600 0.6-0.8, followed by the addition of 0.5 mM IPTG to induce sfGFP expression at 18 °C. After 10 hours, 0.25% L-arabinose was added to activate the autolytic system. The total fluorescence intensity was measured after 16 hours of expression of the induced autolysis system, and after centrifugation, the fluorescence intensity of the supernatant was measured too. The ratio of the fluorescence intensity of the culture and supernatant was used to assess the lysis efficiency of FLSA system.
By comparing with the control group (Figure 3), we determined that the release efficiency of the original FLSA system (FhuD-GGG linker-T7 lysozyme 119V-SsrA) was higher than that of the control group, indicating that the system functioned indeed.
Reference
1. D. Tullman-Ercek et al., Export pathway selectivity of escherichia coli twin arginine translocation signal peptides. J Biol Chem 282, 8309-8316 (2007).
2. F. Zhang et al., N-terminal fused signal peptide prompted extracellular production of a bacillus-derived alkaline and thermo stable xylanase in e. Coli through cell autolysis. Appl Biochem Biotechnol 192, 339-352 (2020).
3. J. Yun, J. Park, N. Park, S. Kang, S. Ryu, Development of a novel vector system for programmed cell lysis in escherichia coli. J Microbiol Biotechnol 17, 1162-1168 (2007).
4. F. W. Studier, Use of bacteriophage t7 lysozyme to improve an inducible t7 expression system. J Mol Biol 219, 37-44 (1991). 5. SnapGene.). Plyss. https://www.snapgene.com/plasmids/pet_and_duet_vectors_(novagen)/pLysS.
6. uniprot.). P00806 · enlys_bpt7. https://www.uniprot.org/uniprotkb/P00806/entry.
7. Q. Chai, Z. Wang, S. R. Webb, R. E. Dutch, Y. Wei, The ssra-tag facilitated degradation of an integral membrane protein. Biochemistry 55, 2301-2304 (2016).
8. F. Zhang et al., Development of a bacterial fhud-lysozyme-ssra mediated autolytic (flsa) system for effective release of intracellular products. ACS Synth Biol 12, 196-202 (2023).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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