In-vitro Phosphorylation of Recombinant Human Tim23 and its Fragments: Post Translational Modification

Author: Chandra Shekhar Anugula, Mir Zahoor Gul and Karuna Rupula

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Abstract

The present study aimed to investigate the post-translational modification of recombinant human Tim23 and its fragments. Recombinant human TIM23 fragments were cloned into the pET28a vector and analyzed by agarose gel electrophoresis. Overexpression of the proteins and their fragments was achieved in BL21DE3 cells followed by affinity purification and validated by SDS-PAGE. In-vitro phosphorylation of the purified proteins was carried out using mitochondrial extract as a kinase source. A specific band corresponding to recombinant human Tim23 after phosphorylation followed by immunoblotting confirmed the phosphorylation of human Tim23. Molecular cloning of human TIM23 was confirmed by the detection of specific bands (gene sizes: 219 bp, 288 bp, 435 bp, and 582 bp) on an agarose gel. Overexpression, purification, and phosphorylation were confirmed by single discrete bands (fragment 2: ~12 kDa, fragment 3: ~18 kDa, and fragment 4: ~22 kDa) on SDS-PAGE. The study highlights the use of indigenously designed primers in the amplification, of Tim23. Further, in the phosphorylation studies, rat liver mitochondrial extract was used as kinase source and Tim23 proteoliposomes were employed to mimic the membrane environment. Overall, the present investigation reports the phosphorylation of Tim23 protein and its fragments under in vitro conditions which may influence the protein transport mechanism and add to the literature on mitochondrial biogenesis.

Keywords

Mitochondria, Recombinant protein, Cloning, Phosphorylation, Tim23, Anti rhuman Tim23

Conclusion

The Autoradiography studies using radiolabelled (γ32P) ATP showed the phosphorylation of full-length human Tim23 and confirmed by western blotting. The amplification of these fragments was evaluated on electrophoresis containing agarose gel (1%). Following digestion (double) with restriction enzymes and agarose gel (1%) electrophoresis revealed the particular insertions of the TIM23 fragments that had been cloned and inserted into the bacterial pET28a vector. Tim23 fragments (f2, f3, and f4) were induced and over expressed in BL21 (DE3) cells by IPTG. The over-expression of proteins was evaluated on SDS-PAGE. The presence of a single distinct band indicated that the r human Tim23 fragments have been purified using affinity chromatography (Ni-NTA), and this has been validated using sodium dodecyl sulphate polyacrylamide gel electrophoresis. The in-vitro phosphorylation of recombinant human Tim23 fragments, as well as the full length of the protein, was shown by autoradiography and verified by immunoblotting using an antibody specific to Tim23. Overall, the present study reports the in-vitro phosphorylation of full-length human Tim23 and its fragments.

References

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How to cite this article

Chandra Shekhar Anugula, Mir Zahoor Gul and Karuna Rupula (2023). In-vitro Phosphorylation of Recombinant Human Tim23 and its Fragments: Post Translational Modification. Biological Forum – An International Journal, 15(2): 665-675.