ABSTRACT
Background: Leishmania major LmSTI1 is a conserved protein among different species of leishmania, and expressed in both amastigote and promastigote forms of L. major life cycle. It has previously been expressed in bacterial systems
Materials and Methods: To express LmSTI1 in the methylotrophic yeast Pichia pastoris [P. pastoris], the shuttle vector pPICZA containing gene lmsti1 was constructed under the control of the AOX1 promoter. The recombinant vector was electro-transformed into P. pastoris, and induced by 0.5% methanol in the buffered medium. The expression of the LmSTI1 protein was visualized in the total soluble protein of P. pastoris by 12% SDS-PAGE, and further confirmed by Western blotting with L.major-infected mouse sera and HRP-conjugated goat anti-mouse IgG as the first and secondary antibodies, respectively
Results: The expression level was 0.2% of total soluble proteins
Conclusion: It might be possible to use this formulation as a whole yeast candidate vaccine against cutaneous leishmanization
ABSTRACT
Expression of eukaryotic proteins in E. coli often results in their aggregation. Proper folding and solubility of therapeutical proteins are the pre-requisite for their bioactivity. This is not achieved in cytoplasmic expression in E. coli because of the absence of disulfide bonds formation. A novel expression/secretion vector was constructed which exploited beta -lactamase signal sequence to translocate processed and soluble proteins into the periplasm of cells. Secretion of model proteins, beta -lactamase and human Epidermal Growth Factor [hEGF] in M15/pSB and M15/pSE systems respectively, was confirmed by SDS-PAGE analysis and bioactivity assay. Secreted hEGF was found to be identical to authentic protein, in size, N-terminal amino acid sequence, biological activity and Western-blotting. The radioimmunoassay revealed 10-fold-higher level of hEGF expression in M15/pSE secretion system compared to that of cytoplasmic expression of the protein. The properly processed and in vivo folded hEGF demonstrated high solubility and bioactivity. The data obtained, evidenced in favor of M15/pSE expression system, which might be suitable to produce the small eukaryotic disulfide-bonded proteins for therapeutical applications or structural studies