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Research and Reviews : A Journal of Life Sciences Original Research

Application of pET28a Vector for the Over Expression of Recombinant Human Tim23 Protein in E. coli and its Validation by Immunoblotting

  1. Chandra Shekhar Anugula1 Department of Biochemistry University College of Science Osmania University, Hyderabad
  2. Karuna Rupula Department of Biochemistry University College of Science Osmania University, Hyderabad

Abstract

This study aimed to produce recombinant human Tim23 protein by molecular cloning, expression, and purification. Amplification of human TIM23 was carried out by polymerase chain reaction (PCR) and cloned into DH5α cells. The pET28a-human TIM23 was transformed into BL21DE3 and expressed the gene by IPTG induction. Recombinant pure human Tim23 antigen was prepared, and injected into rabbits and polyclonal antibodies were raised, which was confirmed by western blotting. The transformed DH5α colonies screened for recombinant TIM23 on agarose gel electrophoresis depicted a specific band corresponding to ~5.4kb pET28a vector and a band at ~0.63 kb confirmed the clone. Overexpression of recombinant human Tim23 was confirmed by SDS-PAGE in BL21DE3 bacterial cells followed by purification by affinity chromatography. Furthermore, polyclonal antibodies were raised and validated by immunoblotting. This study concludes a fundamental and comprehensive study on the molecular cloning, expression, purification, and raising of antibodies against recombinant human Tim23. Characterization of the Tim23 protein will further enhance the existing knowledge on the biogenesis of mitochondria. The polyclonal antibody of Tim23 could be a useful tool for validating regulatory mechanisms in mitochondria.

Keywords

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