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.

中文翻译：

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Cry1Ia信号肽可以改善大肠杆菌和苏云金芽孢杆菌中eGFP或mCherry的表达，并增强宿主的荧光强度。

背景技术分泌蛋白的信号肽（SP）经常被使用或修饰以将重组蛋白引导到原核细胞的细胞质之外。在周质空间和细胞外环境中，重组蛋白远离细胞内蛋白酶，通常它们可以正确有效地折叠。因此，通过SP的存在可以提高重组蛋白的表达水平。但是，很少有人注意将易位效率低的SP用于重组蛋白生产。在本文中，使用荧光蛋白作为报告基因，研究了苏云金芽孢杆菌（Bt）Cry1Ia毒素（Iasp）的信号肽对蛋白表达的调节作用。结果当融合到eGFP或mCherry的N端时，Iasp可以改善荧光蛋白的表达，从而增强大肠杆菌和Bt宿主细胞的荧光强度。实时定量PCR分析显示，在大肠杆菌TG1细胞中，Iegfp的转录水平高于egfp基因的水平。通过免疫印迹分析和共聚焦显微镜观察，证明了IeGFP的转运效率较低。然后使用新型荧光融合蛋白IeGFP比较Bt菌株中cry1Ia（Pi）和cry1Ac（Pac）基因启动子的相对强度，证明后者更强。相比之下，eGFP报告基因不能在相同的敏感性水平下明确指示Pi的调控模式。E的荧光信号。表达Iasp融合mCherry（ImCherry）的大肠杆菌和Bt细胞也得到增强。重要的是，Iasp还可以增强大肠杆菌BL21-star（DE3）菌株中两种难以表达的蛋白质的表达，基质金属蛋白酶13（MMP13）和肌生长抑制素（生长分化因子8，GDF8）。结论我们确定了弱信号肽Iasp对细菌中荧光蛋白和其他重组蛋白表达的积极影响。产生的IeGFP和ImCherry可用作原核细胞中的新型荧光蛋白变体。结果表明Iasp作为一种新型的融合标签，可用于改善重组蛋白表达的潜在应用。大肠杆菌BL21-star（DE3）菌株中的基质金属蛋白酶13（MMP13）和肌生长抑制素（生长分化因子8，GDF8）。结论我们确定了弱信号肽Iasp对细菌中荧光蛋白和其他重组蛋白表达的积极影响。产生的IeGFP和ImCherry可用作原核细胞中的新型荧光蛋白变体。结果表明Iasp作为一种新型的融合标签，可用于改善重组蛋白表达的潜在应用。大肠杆菌BL21-star（DE3）菌株中的基质金属蛋白酶13（MMP13）和肌生长抑制素（生长分化因子8，GDF8）。结论我们确定了弱信号肽Iasp对细菌中荧光蛋白和其他重组蛋白表达的积极影响。产生的IeGFP和ImCherry可用作原核细胞中的新型荧光蛋白变体。结果表明Iasp作为一种新型的融合标签，可用于改善重组蛋白表达的潜在应用。产生的IeGFP和ImCherry可用作原核细胞中的新型荧光蛋白变体。结果表明Iasp作为一种新型的融合标签，可用于改善重组蛋白表达的潜在应用。产生的IeGFP和ImCherry可用作原核细胞中的新型荧光蛋白变体。结果表明Iasp作为一种新型的融合标签，可用于改善重组蛋白表达的潜在应用。