Part:BBa_K5136054

TorA-His tag-linker-sfgfp

Biology

The TorA signal peptide plays a crucial role in the translocation of folded proteins across the membrane via the Tat pathway. Unlike the Sec pathway, which translocates proteins in an unfolded state, the Tat pathway is distinguished by its ability to transport fully folded proteins, often containing complex cofactors, across the membrane. This system is composed of three core components: TatA, TatB and TatC, which together form a translocase capable of accommodating large, structured substrates while maintaining membrane integrity (1).

Usage and Design

The ability of TorA to transport folded proteins makes it a vital tool for recombinant protein secretion. To characterize its secretion efficiency, the TorA signal peptide was fused to the N-terminus of sfGFP via a flexible linker (GGGGS)₃. This fusion was then expressed under the control of the T7 promoter on the pET-28a(+) vector in E. coli BL21(DE3).

Figure 1 Genetic circuit of TorA signal peptide.

Characterization

Agarose Gel Electrophoresis (AGE)

When constructing this circuit of composite part BBa_K5136054, colony PCR and gene sequencing were used to verify that the construction was correct. Target bands (1155 bp) can be observed at the position between 1000 and 2000 bp (Figure 2).

Figure 2 DNA gel electrophoresis of the colony PCR products of BBa_K5136054_pET-28a(+).

Comparative Tests of Performance of Multiple Signal Peptides

The LMT sequence has been identified as a signal peptide capable of directing recombinant proteins out of bacterial cells (XMU-China 2021). Given the variety of engineered signal peptides and corresponding translocation systems developed for secretory production of heterologous proteins in E. coli, we sought to compare the performance of the LMT sequence against other commonly used signal peptides. Specifically, the signal peptides we compared are AIgen, HlyA, OmpA, PelB, TorA, YebF, OsmY and LMT.

All signal peptide-sfGFP fusions were expressed under the control of the T7 promoter on the pET-28a(+) vector in E. coli BL21(DE3) for parallel testing. Following induction, we monitored the fluorescence intensity of the culture, the supernatant after centrifugation, and the OD₆₀₀ of each group over time. Secretion efficiency was calculated at each sampling point as the ratio of fluorescence intensity of supernatant to culture.

Figure 3 Comparative tests of performance of multiple signal peptides. (A) Secretion efficiencies of different groups were calculated as time progressed. (B) The OD₆₀₀, fluorescence intensity of supernatant and normalized culture fluorescence intensity of different groups after induction for 10 hours.

Our results revealed that both OsmY- and HlyA-mediated secretion, with the assistance of HlyB and HlyD, exhibited higher secretion efficiencies than any other signal peptides, including the newly discovered LMT sequence, which ranked third. YebF also performed well in the comparison (Figure 3A). Most of the other signal peptides exhibited secretion efficiencies below 2.5%. These findings demonstrate the variability in secretion efficiencies among different signal peptides, despite the long-term usage of some of them (2).

Interestingly, certain groups showed lower calculated secretion efficiencies than the sfGFP control group (which lacked a signal peptide, No SP). This may be due to the rapid growth and high levels of cytoplasmic sfGFP expression driven by the strong T7 promoter in E. coli BL21(DE3) (3). The normalized fluorescence intensity of the sfGFP-only (No SP) culture was much higher than that of sfGFP fusions with signal peptides, suggesting that even minor leakage due to cell growth or lysis contributed significantly to the fluorescence intensity in the supernatant (Figure 3B). In the case of the LMT signal peptide, despite producing only half the total sfGFP as the no-signal-peptide group (No SP), the fluorescence intensity in the supernatant was comparable to that of the control group and significantly higher than that of any other signal peptide after 10 hours of induction. This highlights the LMT sequence′s efficiency in exporting recombinant proteins out of the bacterial cells.

While OsmY- and HlyA-mediated secretion showed the highest secretion efficiencies, they also impaired cell growth (reflected by the lowest OD₆₀₀ values) and reduced the normalized fluorescence intensity in the culture (Figure 3B). This indicates that overexpression of these systems places a metabolic burden on the host cells, possibly explaining the larger error bars associated with their calculated secretion efficiencies (Figure 3A). Additionally, the normalized fluorescence intensities in all groups with signal peptides were reduced (Figure 3B), potentially due to adverse effects on sfGFP folding caused by the fusion with signal peptides (4).

Classic signal peptides such as PelB and TorA, which direct recombinant proteins to the periplasmic space, exhibited relatively low efficiency for extracellular translocation (Figure 3A) (5). Similarly, OmpA, another well-known signal peptide, showed poor efficiency for recombinant protein secretion, unlike YebF and OsmY, which also mediate extracellular expression (6). Notably, the artificial intelligence-generated signal peptide AIgen, which functions efficiently in Bacillus subtilis, performed poorly in exporting recombinant proteins from E. coli (Figure 3A) (7). However, the OD₆₀₀ values for the AIgen group suggested no significant growth burden (Figure 3B).

In summary, our self-discovered LMT signal peptide showed a slight growth defect but achieved considerable secretion efficiency for the extracellular expression of recombinant proteins, making it a competitive alternative compared to other commonly used signal peptides in E. coli.

Reference

1. B. Sommer et al., Extracellular production and affinity purification of recombinant proteins with Escherichia coli using the versatility of the maltose binding protein. J Biotechnol 140, 194-202 (2009)

2. R. Freudl, Signal Peptides for Recombinant Protein Secretion in Bacterial Expression Systems. Microb. Cell Fact. 17, 52 (2018).

3. S.-I. Tan, I.-S. Ng, New Insight into Plasmid-driven T7 RNA Polymerase in Escherichia coli and Use as a Genetic Amplifier for a Biosensor. ACS Synth. Biol. 9, 613–622 (2020).

4. B. J. Feilmeier, G. Iseminger, D. Schroeder, H. Webber, G. J. Phillips, Green Fluorescent Protein Functions as a Reporter for Protein Localization in Escherichia coli. J. Bacteriol. 182, 4068–4076 (2000).

5. F. J. M. Mergulhão, D. K. Summers, G. A. Monteiro, Recombinant Protein Secretion in Escherichia coli. Biotechnol. Adv. 23, 177–202 (2005).

6. J. T. Boock, D. Waraho-Zhmayev, D. Mizrachi, M. P. DeLisa, “Beyond the Cytoplasm of Escherichia coli: Localizing Recombinant Proteins Where You Want Them” in Insoluble Proteins, E. García-Fruitós, Ed. (Springer New York, New York, NY, 2015; http://link.springer.com/10.1007/978-1-4939-2205-5_5)vol. 1258 of Methods in Molecular Biology, pp. 79–97.

7. Z. Wu, K. K. Yang, M. J. Liszka, A. Lee, A. Batzilla, D. Wernick, D. P. Weiner, F. H. Arnold, Signal Peptides Generated by Attention-based Neural Networks. ACS Synth. Biol. 9, 2154–2161 (2020).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 205