The signal peptide of Cry1Ia can improve the expression of eGFP or mCherry in Escherichia coli and Bacillus thuringiensis and enhance the host's fluorescent intensity
BACKGROUND: The signal peptides (SPs) of secretory proteins are frequently used or modified to guide recombinant proteins outside the cytoplasm of prokaryotic cells. In the periplasmic space and extracellular environment, recombinant proteins are kept away from the intracellular proteases and often they can fold correctly and efficiently. Consequently, expression levels of the recombinant protein can be enhanced by the presence of a SP. However, little attention has been paid to the use of SPs with low translocation efficiency for recombinant protein production. In this paper, the function of the signal peptide of Bacillus thuringiensis (Bt) Cry1Ia toxin (Iasp), which is speculated to be a weak translocation signal, on regulation of protein expression was investigated using fluorescent proteins as reporters.
RESULTS: When fused to the N-terminal of eGFP or mCherry, the Iasp can improve the expression of the fluorescent proteins and as a consequence enhance the fluorescent intensity of both Escherichia coli and Bt host cells. Real-time quantitative PCR analysis revealed the higher transcript levels of Iegfp over those of egfp gene in E. coli TG1 cells. By immunoblot analysis and confocal microscope observation, lower translocation efficiency of IeGFP was demonstrated. The novel fluorescent fusion protein IeGFP was then used to compare the relative strengths of cry1Ia (Pi) and cry1Ac (Pac) gene promoters in Bt strain, the latter promoter proving the stronger. The eGFP reporter, by contrast, cannot indicate unambiguously the regulation pattern of Pi at the same level of sensitivity. The fluorescent signals of E. coli and Bt cells expressing the Iasp fused mCherry (ImCherry) were also enhanced. Importantly, the Iasp can also enhanced the expression of two difficult-to-express proteins, matrix metalloprotease-13 (MMP13) and myostatin (growth differentiating factor-8, GDF8) in E. coli BL21-star (DE3) strain.
CONCLUSIONS: We identified the positive effects of a weak signal peptide, Iasp, on the expression of fluorescent proteins and other recombinant proteins in bacteria. The produced IeGFP and ImCherry can be used as novel fluorescent protein variants in prokaryotic cells. The results suggested the potential application of Iasp as a novel fusion tag for improving the recombinant protein expression.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:19 |
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| Enthalten in: |
Microbial cell factories - 19(2020), 1 vom: 24. Mai, Seite 112 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Gao, Jianhua [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 28.01.2021 Date Revised 28.03.2024 published: Electronic Citation Status MEDLINE |
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| doi: |
10.1186/s12934-020-01371-8 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM310304490 |
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| 100 | 1 | |a Gao, Jianhua |e verfasserin |4 aut | |
| 245 | 1 | 4 | |a The signal peptide of Cry1Ia can improve the expression of eGFP or mCherry in Escherichia coli and Bacillus thuringiensis and enhance the host's fluorescent intensity |
| 264 | 1 | |c 2020 | |
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| 500 | |a Date Completed 28.01.2021 | ||
| 500 | |a Date Revised 28.03.2024 | ||
| 500 | |a published: Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a BACKGROUND: The signal peptides (SPs) of secretory proteins are frequently used or modified to guide recombinant proteins outside the cytoplasm of prokaryotic cells. In the periplasmic space and extracellular environment, recombinant proteins are kept away from the intracellular proteases and often they can fold correctly and efficiently. Consequently, expression levels of the recombinant protein can be enhanced by the presence of a SP. However, little attention has been paid to the use of SPs with low translocation efficiency for recombinant protein production. In this paper, the function of the signal peptide of Bacillus thuringiensis (Bt) Cry1Ia toxin (Iasp), which is speculated to be a weak translocation signal, on regulation of protein expression was investigated using fluorescent proteins as reporters | ||
| 520 | |a RESULTS: When fused to the N-terminal of eGFP or mCherry, the Iasp can improve the expression of the fluorescent proteins and as a consequence enhance the fluorescent intensity of both Escherichia coli and Bt host cells. Real-time quantitative PCR analysis revealed the higher transcript levels of Iegfp over those of egfp gene in E. coli TG1 cells. By immunoblot analysis and confocal microscope observation, lower translocation efficiency of IeGFP was demonstrated. The novel fluorescent fusion protein IeGFP was then used to compare the relative strengths of cry1Ia (Pi) and cry1Ac (Pac) gene promoters in Bt strain, the latter promoter proving the stronger. The eGFP reporter, by contrast, cannot indicate unambiguously the regulation pattern of Pi at the same level of sensitivity. The fluorescent signals of E. coli and Bt cells expressing the Iasp fused mCherry (ImCherry) were also enhanced. Importantly, the Iasp can also enhanced the expression of two difficult-to-express proteins, matrix metalloprotease-13 (MMP13) and myostatin (growth differentiating factor-8, GDF8) in E. coli BL21-star (DE3) strain | ||
| 520 | |a CONCLUSIONS: We identified the positive effects of a weak signal peptide, Iasp, on the expression of fluorescent proteins and other recombinant proteins in bacteria. The produced IeGFP and ImCherry can be used as novel fluorescent protein variants in prokaryotic cells. The results suggested the potential application of Iasp as a novel fusion tag for improving the recombinant protein expression | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Cry1Ia | |
| 650 | 4 | |a Expression level | |
| 650 | 4 | |a Fluorescent intensity | |
| 650 | 4 | |a Fluorescent proteins | |
| 650 | 4 | |a Fusion tag | |
| 650 | 4 | |a Signal peptide | |
| 650 | 7 | |a Bacillus thuringiensis Toxins |2 NLM | |
| 650 | 7 | |a Bacterial Proteins |2 NLM | |
| 650 | 7 | |a Endotoxins |2 NLM | |
| 650 | 7 | |a Hemolysin Proteins |2 NLM | |
| 650 | 7 | |a Luminescent Proteins |2 NLM | |
| 650 | 7 | |a Protein Sorting Signals |2 NLM | |
| 650 | 7 | |a Recombinant Fusion Proteins |2 NLM | |
| 650 | 7 | |a insecticidal crystal protein, Bacillus Thuringiensis |2 NLM | |
| 650 | 7 | |a Green Fluorescent Proteins |2 NLM | |
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| 700 | 1 | |a Qian, Hongmei |e verfasserin |4 aut | |
| 700 | 1 | |a Guo, Xiaoqin |e verfasserin |4 aut | |
| 700 | 1 | |a Mi, Yi |e verfasserin |4 aut | |
| 700 | 1 | |a Guo, Junpei |e verfasserin |4 aut | |
| 700 | 1 | |a Zhao, Juanli |e verfasserin |4 aut | |
| 700 | 1 | |a Xu, Chao |e verfasserin |4 aut | |
| 700 | 1 | |a Zheng, Ting |e verfasserin |4 aut | |
| 700 | 1 | |a Duan, Ming |e verfasserin |4 aut | |
| 700 | 1 | |a Tang, Zhongwei |e verfasserin |4 aut | |
| 700 | 1 | |a Lin, Chaoyang |e verfasserin |4 aut | |
| 700 | 1 | |a Shen, Zhicheng |e verfasserin |4 aut | |
| 700 | 1 | |a Jiang, Yiwei |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Xingchun |e verfasserin |4 aut | |
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