Monday, April 19, 2010
12-37
High-level expression and optimization of 648 Antigen from Leishmania chagasi
Control of human visceral leishmaniasis where the disease is endemic is hampered in part by the limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Humans naturally infected with L.chagasi develop both cellular and antibody responses specific to parasite proteins. Thus, preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines against leishmaniasis. Therefore, in this study, the gene encoding the antigen 648 was cloned into the pQE-30 expression vector in frame with an N-terminal His6 tag under control of the lacO operator, in order to induce high production of recombinant protein. Culture conditions, including the two culture media used (2xTY and TB), pH and the presence IPTG as an inducer, were all optimized in order to obtain an increase of the recombinant protein expression. Results obtained by celullar growth kinetics showed that TB medium showed an increase of approximately 73% for final biomass concentration (based on cell dry weight) compared to 2xTY in this case biomass concentrations were 5.40 g.L-1 and 3.12 g.L-1, respectively. The best productivity results were reached after 1 hour of cultivation, at the instant of culture induction. Medium 2xTY showed a better productivity value (0.0954 g.L-1 h-1) when compared to TB medium value (0.0306 g.L-1 h-1). The protein size was 24 kDa as estimated by SDS-PAGE, and it was purified by a batch mode by IMAC (immobilized metal affinity chromatography) process.
Keywords expression optimization, visceral leishmaniasis, Leishmania chagasi
Keywords expression optimization, visceral leishmaniasis, Leishmania chagasi
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See more of The 32nd Symposium on Biotechnology for Fuels and Chemicals (April 19-22, 2010)
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See more of The 32nd Symposium on Biotechnology for Fuels and Chemicals (April 19-22, 2010)