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

Abstract

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 (P_i) and cry1Ac (P_ac) gene promoters in Bt strain, the latter promoter proving the stronger. The eGFP reporter, by contrast, cannot indicate unambiguously the regulation pattern of P_i 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.

Keywords: Cry1Ia; Expression level; Fluorescent intensity; Fluorescent proteins; Fusion tag; Signal peptide.

Fig. 1

The expression cassettes of the fusion fluorescent proteins. The P_ac-pelBegfp-T_ac and P_ac-torAegfp-T_ac expression cassettes are similar to the P_ac-Iegfp-T_ac except the leader sequence at the 5′ flanking of egfp gene. The expression cassettes P_T7-NusA-egfp-T_T7, P_T7-MBP-egfp-T_T7, P_T7-I-mmp13-T_T7, P_T7-Trx-mmp13-T_T7, P_T7-I-gdf8-T_T7 and P_T7-Trx-gdf8-T_T7 share same structures with P_T7-Trx-egfp-T_T7

Fig. 2

Expression analysis of eGFP and IeGFP regulated by P_ac promoter in E.coli TG1 strain. a Lane “-” is the negative control which prepared from T304 cells sampled at 12 h after inoculation. TAc-IeGFP and TAc-eGFP cells were separately taken at 4, 8, 10, 12 and 24 h after inoculation. Lane “M” is the molecular weight standards. b Comparison of the collected cells when they were incubated for 24 h. c The fluorescent intensities and their fold changes for TAc-IeGFP over TAc-eGFP strains at corresponding sampling time. The slit widths of EX/EM were 3 nm and 5 nm respectively and the detections were conducted in low sensitivity. The fluorescent signals of T304 cells cannot be detected. The error bars indicate standard error of mean. The significant differences of the fluorescent intensities between TAc-IeGFP and TAc-eGFP cells at corresponding time were indicated by single asterisk (p < 0.05) or double asterisks (p < 0.01)

Fig. 3

Expression analysis of eGFP and IeGFP regulated by the P_ac promoter in Bt strain. a, b The expression of IeGFP (panel a) and eGFP (panel b) in Bt at different times (9, 12, 24, 36, 48, 60 and 72 h after inoculation) were analyzed respectively by western blot. “S” represents the supernatant of cell culture and “P” represents the resuspended cells by PBS buffer. Lane “M” is the molecular weight standards. c, d The fluorescent intensities of the harvested cells (panel c) or the supernatants of cell cultures (panel d) and their fold changes for BAc-IeGFP over BAc-eGFP strain at corresponding culture time. The slit widths of EX/EM were both 3 nm and the detections were conducted in low sensitivity for the resuspended cells and in high sensitivity for the supernatant. The fluorescent signals of B304 cells cannot be detected and the fluorescent intensity of the 72 h supernatant of BAc-IeGFP strain cell culture was beyond the limit (1000 A.U.). The error bars indicate standard error of mean. The significant differences of the fluorescent intensities between BAc-IeGFP and BAc-eGFP strains at corresponding time were indicated by single asterisk (p < 0.05) or double asterisks (p < 0.01). e Comparison of the collected cells before (up) and after excitement (down) when they were incubated for 24 h and 48 h. The cells were excited by blue light using Luyor 3415RG hand held lamp

Fig. 4

The expression analysis of IeGFP in E. coli BL21-star (DE3). IeGFP (lane “I”) and eGFP (lane “e”) expressed in E. coli BL21-star (DE3) at the different induction temperatures were analyzed by SDS-PAGE. Lane “-” is the negative controls sampled from the BL28aD cell culture and lane “M” is the molecular weight standards. The arrow indicates the IeGFP expressed at 16 °C

Fig. 5

Prediction of the secondary structure of sequences near the initial codon. The secondary structure of sequences near the initial codon (−4 ~ + 37) of egfp (left) and Iegfp (right) gene were predicted by RNAstructure software. The initial codon is indicated in red. The folding energies (dG) are also indicated respectively

Fig. 6

Expression analysis of the different fluorescent proteins in E. coli strain. a The expression of eGFP (lane “e”), IeGFP (lane “Ie”), eGFP-I (lane “eI”), pelB-eGFP (lane “pe”) and torA-eGFP (lane “te”) at different times (4, 8, 10, 12 and 24 h after inoculation) were analyzed by western blot. Lane “-” is the negative control which prepared from T304 cells sampled at the corresponding times respectively. Lane “M” is the molecular weight standards. The arrows indicate the expressed IeGFP at different times. b The optical densities at 600 nm (OD₆₀₀) of the E. coli strains expressing the different fluorescent proteins were monitored during the first 6 h after inoculation. The error bars indicate standard deviation of mean. c The fluorescent intensities of the samples obtained at corresponding times in panel a (slit widths of EX/EM = 3/5 nm in low sensitivity). The fluorescent signals of T304 cells cannot be detected. The error bars indicate standard error of mean

Fig. 7

Confocal images analysis of E. coli cells expressing different fluorescent proteins. The E. coli cells expressing eGFP (d–f), IeGFP (g–i), eGFP-I (j–l), torA-eGFP (m–o) or pelB-eGFP (p–r) were observed by inverted confocal microscope (Leica SP8). The cells of T304 strain (a–c) were used as the negative control. The bright-field images and the fluorescent images were arranged in the first and second row, respectively, and then were merged (the third row). The scale bars represent 5 μm

Fig. 8

Fluorescent intensities analysis of the P_i promoter regulated eGFP and IeGFP in Bt strain. The fluorescent intensities of the resuspended cells by PBS buffer (a) and the supernatants of cell cultures (b) were detected respectively. The samples were obtained at different times (9, 12, 24, 36, 48, 60 and 72 h after inoculation). The slit widths of EX/EM were 3/3 nm and the detections were conducted in high sensitivity. The fluorescent signals of B304 cells cannot be detected and the fluorescent intensity of the 36 h cells of BI-IeGFP strain was beyond the limit (1000 A.U.). The significant difference of the fluorescent intensities between BI-IeGFP and BI-eGFP strains at corresponding time were indicated by single asterisk (p < 0.05). The error bars indicate standard error of mean

Fig. 9

Expression analysis of mCherry and ImCherry in E. coli TG1 and Bt BMB171 strain. a The fluorescent intensities of the harvested TG1 cells expressing mCherry or ImCherry were monitored at different times (4, 8, 10, 12 and 24 h after inoculation). The slit widths of EX/EM were 1.5 nm and 3 nm, and the detections were conducted in high sensitivity. The fluorescent signals of B304 cells cannot be detected. The error bars indicate standard error of mean. The significant differences of the fluorescent intensities between TAc-ImCherry and TAc-mCherry cells at corresponding time were indicated by single asterisk (p < 0.05) or double asterisks (p < 0.01). b Comparison of the collected E. coli TG1 cells when they were incubated for 24 h. c Western blot analysis of the expression of mCherry and ImCherry in E. coli TG1 strain. “-” lane is negative control which prepared from T304 cells sampled at 12 h after inoculation. TAcImCherry and TAcmCherry cells were separately taken at 4, 8, 10, 12 and 24 h after inoculation. “M” represents the molecular weight standards. d, e The fluorescent intensities of the supernatants of cell cultures (panel D, slit widths of EX/EM = 3/5 nm in high sensitivity) or the harvested Bt cells (e slit widths of EX/EM = 3/3 nm in high sensitivity) at corresponding culture time. The fluorescent signals of B304 cells cannot be detected. The error bars indicate standard error of mean. The significant differences of the fluorescent intensities between BAc-ImCherry and BAc-mCherry cells at corresponding time were indicated by double asterisks (p < 0.01). f Western blot analysis of the expression of ImCherry in Bt BMB171 strain. “S” represents the supernatant of cell culture and the “P” means the resuspended cells by PBS buffer. “M” represents the molecular weight standards