Analysis of the Mycobacterium protein Cpn60.1 based on chimaeras expression in Escherichia coli

Abstract

Chaperonins are proteins that facilitate protein folding but might have other roles. Zebrafish is the model used to study the novel functions of chaperonin proteins infected with Mycobacterium marinum rather than Mycobacterium tuberculosis, the leading cause of tuberculosis. The Cpn60.1 protein expression in the double phosphomimetic mutation (DPM) of M. marinum in Escherichia coli did not complement the loss of GroEL. Fragments swapping between C-terminals of M. marinum and M. tuberculosis was performed to provide the constructed chimaeras that might be useful for determining whether the Cpn60.1 protein of the mutated M. marinum will complement the absence of GroEL in Escherichia coli. Complementation assays were performed in this study to validate the result of the previous laboratory work that proved the complementation of the DPM-M. tuberculosis expressed in Escherichia coli. In this study, the site-directed mutagenesis method successfully introduced the HindIII site in M. marinum, which confirmed by Sanger sequencing analysis. A set of experiments were also performed, including DNA ligation, dephosphorylation and transformation, in which fragments were swapped between the HindIII sites of M. marinum and M. tuberculosis. The findings of this study show that neither M. marinum nor M. tuberculosis obtained the required chimaeras. Therefore, the Cpn60.1 proteins of M. marinum are still not complemented when expressed in E. coli.