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Rapid monitoring of the target protein expression with a fluorescent signal based on a dicistronic construct in Escherichia coli.

ABSTRACT: Real-time quantification of recombinant proteins is important in studies on fermentation engineering, cell engineering, etc. Measurement of the expression level of heterologous proteins in bacterial fermentation broth has traditionally relied on time-consuming and labor-intensive procedures, such as polyacrylamide gel electrophoresis, immunoblot analysis, and biological activity assays. We describe a simple, fast, and high sensitive assay for detecting heterologous proteins production in bacteria either at the overall level (fluorescence spectrophotometry) or at the individual level (fluorescence microscopic image) in this study. Based on a dicistronic model, the translation of target gene in the upstream open reading frame (ORF) was coupled with the synthesis of the mCherry reporter in the downstream ORF in E. coli cells, and subsequently this demonstrated a positive correlation between the expression of target gene and mCherry. Although a time lag exists between the expression of target protein and mCherry reporter, the method described here allows facile monitoring of dynamic changes in target protein expression, relying on indirect determination of the fluorescence intensity of mCherry during fermentation in real-time models. Additionally, the performance of a single bacterial cell factory could be checked under the fluorescence microscope field.

SUBMITTER: Hui CY

PROVIDER: S-EPMC5962521 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Rapid monitoring of the target protein expression with a fluorescent signal based on a dicistronic construct in Escherichia coli.

Hui Chang-Ye CY Guo Yan Y Zhang Wen W Huang Xian-Qing XQ

AMB Express 20180521 1

Real-time quantification of recombinant proteins is important in studies on fermentation engineering, cell engineering, etc. Measurement of the expression level of heterologous proteins in bacterial fermentation broth has traditionally relied on time-consuming and labor-intensive procedures, such as polyacrylamide gel electrophoresis, immunoblot analysis, and biological activity assays. We describe a simple, fast, and high sensitive assay for detecting heterologous proteins production in bacteri ...[more]

PMID: 29785487

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Project description: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.

Dataset Information

Rapid monitoring of the target protein expression with a fluorescent signal based on a dicistronic construct in Escherichia coli.

Publications

Rapid monitoring of the target protein expression with a fluorescent signal based on a dicistronic construct in Escherichia coli.

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