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. 2021 Feb 25;11(1):4576.
doi: 10.1038/s41598-021-84188-8.

Comparison of E. coli based self-inducible expression systems containing different human heat shock proteins

Fatemeh Sadat Shariati  1 , Malihe Keramati  1 , Vahideh Valizadeh  1 , Reza Ahangari Cohan  2 , Dariush Norouzian  3
Affiliations

Comparison of E. coli based self-inducible expression systems containing different human heat shock proteins

Fatemeh Sadat Shariati et al. Sci Rep. .

Abstract

IPTG-inducible promoter is popularly used for the expression of recombinant proteins. However, it is not suitable at the industrial scale due to the high cost and toxicity on the producing cells. Recently, a Self-Inducible Expression (SILEX) system has developed to bypass such problems using Hsp70 as an autoinducer. Herein, the effect of other heat shock proteins on the autoinduction of green fluorescent protein (EGFP), romiplostim, and interleukin-2 was investigated. For quantitative measurements, EGFP expression was monitored after double-transformation of pET28a-EGFP and pET21a-(Hsp27/Hsp40/Hsp70) plasmids into E. coli using fluorimetry. Moreover, the expression level, bacterial growth curve, and plasmid and expression stability were compared to an IPTG- inducible system using EGFP. Statistical analysis revealed a significant difference in EGFP expression between autoinducible and IPTG-inducible systems. The expression level was higher in Hsp27 system than Hsp70/Hsp40 systems. However, the highest amount of expression was observed for the inducible system. IPTG-inducible and Hsp70 systems showed more lag-time in the bacterial growth curve than Hsp27/Hsp40 systems. A relatively stable EGFP expression was observed in SILEX systems after several freeze-thaw cycles within 90 days, while, IPTG-inducible system showed a decreasing trend compared to the newly transformed bacteria. Moreover, the inducible system showed more variation in the EGFP expression among different clones than clones obtained by SILEX systems. All designed SILEX systems successfully self-induced the expression of protein models. In conclusion, Hsp27 system could be considered as a suitable autoinducible system for protein expression due to less metabolic burden, lower variation in the expression level, suitable plasmid and expression stability, and a higher expression level.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The intensity of fluorescence signals within 6 h incubation. The average of the fluorescence signal intensity for 10 different clones from SILEX systems was plotted for 6 h incubation. Escherichia coli Bl21 (DE3) containing plasmid pET28a-EGFP was evaluated without adding an inducing agent to observe the basal expression of the EGFP protein (Negative control). Data are represented as Mean ± SD from ten independents clones.
Figure 2
Figure 2
The nonlinear regression (Logistic growth) of bacterial growth curves for the inducible and autoinducible expression systems. Escherichia coli BL21 (DE3) strain containing pET28a-EGFP was evaluated as a control. Data are represented as Mean ± SD form three independent measurements.
Figure 3
Figure 3
The expression level monitoring of transformed bacteria after four freeze-thawing cycles at − 70 °C within 90 days. Escherichia coli Bl21 (DE3) containing plasmid pET28a-EGFP was also evaluated without adding the inducing agent to observe the basal expression of the target protein. Data are represented as Mean ± SD from three independent measurements.
Figure 4
Figure 4
The SDS-PAGE analysis of EGFP expression in inducible and autoinducible systems. (a) Hsp27 SILEX system [lane 1: 2 h after inoculation, Lane 2: 16 h after inoculation, and lane 3: protein marker], (b) Hsp40 SILEX system [lane 1: 2 h after inoculation, Lane 2: 16 h after inoculation, and lane 3: protein marker (kDa)], (c) hsp70 SILEX system [lane 1: 2 h after inoculation, Lane 2: 16 h after inoculation, and lane 3: protein marker], and (d) Inducible system [lane 1: after induction, lane 2: before induction, and lane 3: protein marker]. The green arrow indicates the expression of EGFP and the red arrow indicates the leaky expression of the Hsp27, Hsp40, and Hsp70 on the gel a, b, and c, respectively. The protein marker also shows proteins with the molecular weights of 180, 135, 100, 75, 63, 48, 35, 25, 17, 11 kDa.
Figure 5
Figure 5
The SDS-PAGE analysis of expression of romiplostim and interleukin-2 in the SILEX systems. (a) Romiplostim: Hsp27 SILEX system [lane 2: 2 h after inoculation, Lane 3: 16 h after inoculation], Hsp40 SILEX system [lane 4: 2 h after inoculation, Lane 5: 16 h after inoculation], Hsp70 SILEX system [lane 6: 2 h after inoculation, Lane 7: 16 h after inoculation], and lane 1: protein marker. (b) Interleukin-2: Hsp27 SILEX system [lane 2: 2 h after inoculation, Lane 3: 16 h after inoculation], Hsp40 SILEX system [lane 4: 2 h after inoculation, Lane 5: 16 h after inoculation], Hsp70 SILEX system [lane 6: 2 h after inoculation, Lane 7: 16 h after inoculation], and lane 1: protein marker. The red arrow indicates the expression of romiplostim and interleukin-2 and the green arrow indicates the leaky expression of Hsp27, Hsp40, and Hsp70 on the gel a, b, and c, respectively. The protein marker also shows proteins with the molecular weights of 180, 135, 100, 75, 63, 48, 35, 25, 17, 11 kDa.
Figure 6
Figure 6
Box plot graph of the protein expression levels in the expression systems. As result depicted, a higher variation was observed for the IPTG-inducible system in contrast to the SILEX systems.
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References

    1. Makoff A, Oxer M. High level heterologous expression in E. coli using mutant forms of the lac promoter. Nucleic Acids Res. 1991;19:2417–2421. doi: 10.1093/nar/19.9.2417. - DOI - PMC - PubMed
    1. Riggs PD. Overview of protein expression vectors for E. coli. Curr. Protoc. Essential Lab. Tech. 2018;17:e23. doi: 10.1002/cpet.23. - DOI
    1. Gombert A, Kilikian B. Recombinant gene expression in Escherichia coli cultivation using lactose as inducer. J. Biotechnol. 1998;60:47–54. doi: 10.1016/S0168-1656(97)00185-5. - DOI - PubMed
    1. Menzella HG, Ceccarelli EA, Gramajo HC. Novel Escherichia coli strain allows efficient recombinant protein production using lactose as inducer. Biotechnol. Bioeng. 2003;82:809–817. doi: 10.1002/bit.10630. - DOI - PubMed
    1. Menzella HG, Gramajo HC. Recombinant protein production in high cell density cultures of Escherichia coli with galactose as a gratuitous inducer. Biotechnol. Prog. 2004;20:1263–1266. doi: 10.1021/bp034365+. - DOI - PubMed

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