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

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.

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system

“…The simultaneous expression of heat shock protein with the target protein can rapidly prevent instability and decrease the stress induced by repeated freeze–thaw cycles [ 23 ]. This phenomenon is also in agreement with our previous study on SILEX systems [ 20 ]. Moreover, since heat shock proteins also act as chaperones, the use of bacteria- or human-originated heat shock proteins in these systems can offer better folding of the complex proteins that need further investigations [ 24 – 28 ].…”

“…This approach has the following steps: (1) plating different dilutions of transformants on agar plates; (2) cultivation of single colonies in 96-deep-well microplates; (3) induction of expression by adding IPTG; (4) measuring the fluorescent signals by a fluorimeter; (5) and finally, analyzing the soluble and insoluble fractions with SDS-PAGE and western blot [ 19 ]. In our previous study, we stated that autoinduction by Hsp27 is a convenient way to produce recombinant proteins without IPTG addition [ 20 ]. The developed autoinducible system does not need optical density monitoring, therefore reducing the operator intervention, which is preferable for high-throughput purposes.…”

Design and characterization of a novel lytic protein against Clostridium difficile

“…Interestingly, target proteins were also expressed in E. coli Rosetta cells without IPTG induction; it implied that the leaky expressions existed in these recombinant bacteria. As reported by previous studies, this leaky expression was caused by the low level of cellular β-galactosidase which competitively combined with repressor protein to open the lac operon (Gatti-Lafranconi et al 2013;Shariati et al 2021). After purification by one-step Ni-NTA affinity chromatography, high purities of Cwp19-CD (80.2%), sumo-Acd-CD (93.8%), and sumo-Cwl-CD (70.6%) were obtained (Fig.…”

Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21

“…As has been demonstrated in this study, the negative effects of this imbalance can be minimized by tuning heterologous gene expression through vector copy number-promoter strength balance. Therefore, studies on plasmid copy number combined with the type of replication origin and promoter characteristics give important information to improve synthetic biology in heterologous protein and metabolite production method application (Koma et al, 2018;Shariati et al, 2021). In conclusion, the results show the importance of the transcription…”