Part:BBa_K5292311
OmpT
This signal peptide was selected through model prediction from a library of approximately 200 signal peptides in Escherichia coli, aiming to enhance the secretion expression of the PET-degrading enzyme ICCG. This signal peptide originates from library of signal peptides in Escherichia coli and was initially used to express Outer membrane protein T We constructed the signal peptide at the N-terminus of the ICCG-GFP fusion protein and characterized its secretion efficiency through fluorescence intensity. Although the secretion efficiency of this signal peptide in the experiment was not significantly improved, it provided valuable reference data for further optimization of PET-degrading enzyme expression.
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]
Abstract
This signal peptide was selected through model prediction from a library of approximately 200 signal peptides in Escherichia coli, aiming to enhance the secretion expression of the PET-degrading enzyme ICCG. This signal peptide originates from... and was initially used to express... We constructed the signal peptide at the N-terminus of the ICCG-GFP fusion protein and characterized its secretion efficiency through fluorescence intensity. Although the secretion efficiency of this signal peptide in the experiment was not significantly improved, it provided valuable reference data for further optimization of PET-degrading enzyme expression.
In projects, this signal peptide can be used to guide the secretion expression of target proteins, especially in studies of recombinant protein secretion and applications in synthetic biology. Combining model predictions and experimental data, this signal peptide lays the foundation for subsequent optimization of signal peptides.
Profile
Name：OmpT
Origin:Outer membrane protein T
Properties: Guide the target protein through the inner membrane for secretion, located at the N-terminus of the target protein, assisting in the protein's entry into the periplasmic space or secretion outside the cell.
Usage and Biology
This signal peptide is derived from the Escherichia coli signal peptide library and was selected through model prediction to enhance the secretion expression of target proteins. In the experiment, we constructed this signal peptide at the N-terminus of the ICCG-GFP fusion protein to help guide ICCG protein secretion. By measuring fluorescence intensity, we were able to characterize its impact on protein secretion efficiency. Although the experimental results did not achieve the expected significant improvement, this signal peptide provides valuable data support for further optimization of secretion efficiency. In synthetic biology and recombinant protein expression projects, this signal peptide can be used as a tool to enhance secretion expression, suitable for various protein secretion studies.
This signal peptide is based on the Escherichia coli signal peptide, selected through model prediction for its potential high secretion efficiency. It is located at the N-terminus of the target protein and functions primarily to guide the protein through the inner membrane into the periplasmic space or secrete it outside the cell. The H region of the signal peptide contains key hydrophobic amino acids that effectively bind to the inner membrane and assist in the transmembrane transport of proteins. Although this experiment did not significantly improve the secretion efficiency of ICCG, this signal peptide provides a reference for future optimization of protein secretion pathways and has potential for further refinement.
Design Notes
We used PCR amplification to obtain a linearized vector and seamless cloning to obtain recombinant plasmids carrying the mutated nfaA signal peptide. The recombinant plasmids was then transformed into the Escherichia coli strain DH5α. We then sequenced the amplified recombinant plasmid and obtained the correctly constructed recombinant plasmid.
Characterization
To characterize the activity of the signal peptide, we constructed an ICCG-GFP fusion protein, using fluorescence intensity as a quantitative measure of expression. The following steps were taken to characterize the mutants:
- The constructed MnfaA signal peptide mutant plasmids were transformed into Escherichia coli BL21 (DE3) expression strains.
- The transformed strains were cultured in a high-throughput screening system, and the expression levels of different mutants were compared by measuring fluorescence/OD600.
The high-throughput screening system is as follows:
Induce Temperature (25℃), Induce Time (12 h), Concentration of IPTG (1.0 mM)
1. All the signal peptides and promoters mutants which we had designed were constructed on pET26-ICCG-GFP and were transformed into BL21 (DE3), and were selected for screening.
2. The above BL21 (DE3) strains containing recombinant protein-encoding plasmids were selected and grown in 96-deep-well plates with 600 μL of LB liquid medium with corresponding resistance per well for 6 h at 37 °C.
3. Then, 8 μL of the bacterial culture were transferred into 96-deep-well plates with 800 μL of LB liquid medium per well and grown for 6 h at 37 °C.
4. 100 μL samples were transferred into transparent 96-well microplates for OD600 measurements before the inducer was added.
5. Then, 7 μL inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) for each wells was added to a final concentration of 1.0 mM, and the cultivation was continued at 25℃ for 12 h.
6. Take 100 μL from each well add to test the OD600 before add the inducer.
7. 100 μL samples were transferred into transparent 96-well microplates for OD600 measurements and 100 μL samples were transferred into 96-well black microplates for GFP fluorescence measurement.
8. The fluorescence intensity was measured using an excitation wavelength of 485 nm and an emission wavelength of 520 nm by using EnVision Multilabel Reader.
Reference
Grodberg J, Lundrigan MD, Toledo DL, Mangel WF, Dunn JJ. Complete nucleotide sequence and deduced amino acid sequence of the ompT gene of Escherichia coli K-12. Nucleic Acids Res. 1988 Feb 11;16(3):1209. doi: 10.1093/nar/16.3.1209. PMID: 3278297; PMCID: PMC334759.
Sugimura K, Nishihara T. Purification, characterization, and primary structure of Escherichia coli protease VII with specificity for paired basic residues: identity of protease VII and OmpT. J Bacteriol. 1988 Dec;170(12):5625-32. doi: 10.1128/jb.170.12.5625-5632.1988. PMID: 3056908; PMCID: PMC211661.
Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y. The complete genome sequence of Escherichia coli K-12. Science. 1997 Sep 5;277(5331):1453-62. doi: 10.1126/science.277.5331.1453. PMID: 9278503.
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