Escherichia coli (Migula, 1895) Castellani & Chalmers, 1919

Fesharaki-Esfahani, Monireh, Shahpiri, Azar & Kazemi-Nasab, Akram, 2021, A highly efficient, thermo stable and broad pH adaptable copper-zinc super oxide dismutase (AmSOD 1) mediates hydrogen peroxide tolerance in Avicennia marina, Phytochemistry (112766) 187, pp. 1-9 : 4

3.3. Heterologous expression of His-AmSOD in E. coli

The gene encoding AmSOD1 contains 459 base pairs that encode a protein containing 152 amino acid. Since the gene was cloned in pET28a, the protein was expected to be produced as fusion protein with His.tag. In the present work the strain R-AmSOD1 (Rosetta stain carrying pET28a) was grown in the LB medium with kanamycin and chloramphenicol. The Rosetta strain containing empty pET28a was considered as control strain. Following induction with IPTG, the recombinant protein His-AmSOD1 was expressed in R-AmSOD1. The corresponding band was not observed in the control strain. The theoretical molecular weight for His-AmSOD1 was 19 kDa. SDS-PAGE of cell extracts showed a prominent polypeptide band of the expected molecular mass ( Fig. 3A ). Furthermore, the comparison between the intensity of band corresponding to His-AmSOD 1 in the soluble fraction and the intensity of His-AmSOD1 band in the total fraction indicated that the protein His-AmSOD1 has a high solubility ( Fig. 3B ). Whereas the addition of zinc in the medium was not affected on the solubility of His-AmSOD1, the solubility was increased in the presence of copper. The solubility of protein with supplementation of both zinc and copper was almost similar to solubility with supplementation of copper alone (Supplemental Fig. S2 ).

The recombinant His-AmSOD1 was purified from the soluble fraction using nickel affinity chromatography in yields of 30 mg /L. The quality of purification was assessed by SDS-PAGE analysis ( Fig. 3C ).

Figures

Fig. 3. SDS-PAGE analysis and the solubility of expressed His-AmSOD1 in the strain R-AmSOD1 harboring pET28a-AmSOD1 (A) Total soluble protein extracted from control strain and the strain R-AmSOD1 before and 1–4 h after induction with IPTG. (B) The study of solubility of His-AmSOD1 by comparison of His-AmSOD1 band intensities in the total protein (T), the soluble fraction extracted from R-AmSOD1 (S) and the unsoluble fraction (U) extracted from R- AmSOD1 (The recombinant proteins were shown with arrows) (C) SDS-PAGE analysis of AmSOD1 after purification with affinity chromatography.

Fig. 2. The effects of salt and hydrogen peroxide stress on the appearance pattern and activity of AmSOD1 in the leaves of A. marina (A) Appearance pattern of AmSOD1 and (B) AmSOD assay in the leaves responding to hydrogen peroxide and salt stress as well as control at various time points (0–48 h). Each histogram represents the mean obtained from three independent reactions (± SD). Different letters above the bars indicate statistically significant differences by LSD test.