Abstract
Objective
To obtain a recombinant flagellin derivative CBLB502, expressed in functionally soluble form, the technology of library construction and screening of synonymous codon variants was employed, and its expression, solubility, and activity were assessed.
Results
We screened several synonymous codon variants scvCBLB502s with the enhanced solubility from the constructed library, harboring the random substitutions of the first ten amino acid residues of the parental CBLB502 with synonymous codons. Among them, scvCBLB502-5 was purified (> 8.4 mg/l) by single step procedure using an affinity chromatography without any ancillary treatment with protease inhibitor cocktail solution and/or boiling at 90 °C. Subsequent study showed that the recombinant protein scvCBLB502-5 distinctly induced the TLR5 (Toll-Like Receptor 5)-mediated NF-κB activation and also IL-8 production in HEK293-hTLR5 cells.
Conclusion
Results showed that scvCBLB502-5, engineered through the synonymous codon substitutions, was easily expressed in functionally soluble form and maintained the proper folding to be recognized by TLR5, as an inducer for pathogen-associated molecular pattern (PAMP).
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Acknowledgements
This work was supported by Basic Science Research programs [NRF-2017R1A2B4007852 (to D.-H.L), NRF-2017R1D1A1B03035338 (to D.-E.C)] of the National Research Foundation (NRF) of Korea funded by the Ministry of Education, Science and Technology of Korea (MEST). This work was also supported by the Intelligent Synthetic Biology Center [NRF-2017030616 (to G.-J.K)] program of NRF funded by the Ministry of Science, ICT & Future Planning and supported by a Grant from Marine Biotechnology Program [20170305 (to G.-J.K)] funded by Ministry of Oceans and Fisheries, Korea.
Supporting information
Supplementary Fig. 1 Expression pattern of opt-CBLB502 under T7 promoter in E. coli BL21 (DE3). T and S means total and soluble fraction, respectively. Red arrow indicates opt-CBLB502. M, protein size marker.
Supplementary Fig. 2 Codon substitutions in the 5′-coding region of each selected variants.
Supplementary Fig. 3 The relative fluorescence intensity of mCherry for each mCherry-fused variant. Fluorescence intensity of 12 screened scvCBLB502s was assessed with a microplate reader.
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G-JK and D-HL designed research, analyzed the data, and wrote the paper. D-EC designed research, performed the experiments, analyzed the data, and wrote the paper. J-HL performed the research experiments and analyzed the data. H-JC performed the research experiments and analyzed the data. S-KY performed the research experiments and analyzed the data.
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Cheong, DE., Lee, J., Choi, HJ. et al. Soluble overexpression of a flagellin derivative from Salmonella enterica using synonymous codon substitutions of 5′-coding region in Escherichia coli. Biotechnol Lett 41, 1275–1282 (2019). https://doi.org/10.1007/s10529-019-02733-y
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DOI: https://doi.org/10.1007/s10529-019-02733-y