Expression and preliminary characterization of the potential vaccine candidate LipL32 of leptospirosis

TLDR: In this paper, the over expression of LipL32 and hGMCSF genes into yeast expression system for obtaining a high yield of recombinant protein production was investigated for leptospirosis.

Abstract: Leptospirosis is a globally re-emerging infectious disease mainly for mammals. The infection is caused by the spirochete Gram-negative bacterium Leptospira interrogans, which affects animals and humans worldwide. In our previous studies, recombinant protein production has been obtained from the bacterial expression system. In this study, we have investigated the over expression of LipL32 and hGMCSF genes into yeast expression system for obtaining a high yield of recombinant protein production. Here, we described the yeast expression studies with several applications such as protein folding, fast growth, and post-translational modification. The expression studies were carried out in a novel protein expression system, the methylotrophic yeast Pichia pastoris KM71 strain. The LipL32, Green fluorescent protein (EGFP), and human granulocyte–macrophage colony-stimulating factor (hGMCSF) genes were cloned into pPIC9 yeast expression vector. The recombinant clones of pPIC9-EGFP-LipL32 and pPIC9-EGFP-hGMCSF-LipL32 were transformed into Pichia pastoris KM71 strain by electroporation. Media optimization and other physiological characters were studied for the transformed recombinant protein. The protein was then purified using a Ni–NTA column; meanwhile, the recombinant DNA constructs contain His-tag at the C- terminal end. Finally, the intracellular EGFP expression of pPIC9-EGFP-LipL32, and pPIC9-EGFP-hGMCSF-LipL32 in Pichia pastoris KM71 strain was confirmed by fluorescence microscopic analysis. Protein–protein dockings were done to study LipL32-Adjuvant (hGMCSF, hIgGFC, and hC3d) interactions. Furthermore, this docking analysis was shown better interaction between LipL32, and hGMCSF, which is also used for the enhanced vaccine potential against leptospirosis.