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Optimization of induction parameters, structure quality assessment by ATR-FTIR and in silico characterization of expressed recombinant polcalcin in three different strains of Escherichia coli.

Afshin Derakhshani,Kamyar Keshavarz F,Sahar Barzegari Banadkoki,F. Shirazi,M. Barati,M. Fereidouni,H. Safarpour

Published 2019 in International Journal of Biological Macromolecules

ABSTRACT

The inhalation of Chenopodium album (C. album) pollen, especially polcalcin (Che a 3), has been reported as a significant source of allergic respiratory symptoms. This study was conducted to characterize biophysical differences of recombinant polcalcin come from three different Escherichia coli strains using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), optimize recombinant polcalcin expression, and linear B-cell epitopes identification using in silico methods. ATR-FTIR results of purified proteins showed spectra intensity variance in the amide I region, while there were no changes in pick position and shape of the bands. SHuffle® T7 Express was selected for subsequent optimization due to ability in the correction of the mis-oxidized bonds and promotes proper folding which was validated by ATR-FTIR analysis results. Then, Response Surface Methodology was performed to optimize critical factors including induction temperature, duration of induction, and concentration of inducer. The best partitioning conditions were achieved in 1.5 mM IPTG for 10.97 h at 29.9 °C. Finally, prediction of polcalcin B-cell epitopes was carried out which indicated the presence of 4 different epitopes. Together, the results may help to the development of diagnostic approaches and also vaccine manufacture for desensitization and modulation of the allergic response in patients.

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