Abstract
BackgroundDengue is a viral disease spread to humans by mosquitoes. Notably, there are four serotypes of dengue viruses (DENV) that places ~40 % of the global population at risk of infection. However, lack of a suitable drug or a preventive vaccine exacerbates the matter further. Envelope domain-III (EDIII) antigen of dengue virus (DENV) has garnered much attention as a promising vaccine candidate for dengue, in addition to its use as a diagnostic intermediate. Hence developing a method for efficient production of high quality recombinant EDIII is important for research and industrial purpose.ResultsIn this work, a Pichia pastoris system was optimized for the secretory over-expression of DENV serotype-3 EDIII under the control of methanol inducible AOX1 promoter. Temperature alone had a significant impact upon the amount of secretory EDIII, with 2.5-fold increase upon reducing the induction temperature from 30 to 20 °C. However surprisingly, supplementation of culture media with Casamino acids (CA), further augmented secretory EDIII titer, with a concomitant drop of intracellular EDIII levels at both temperatures. Though, reduction in intracellular retention of EDIII was more prominent at 20 °C than 30 °C. This suggests that CA supplementation facilitates overexpressing P. pastoris cells to secrete more EDIII by reducing the proportion retained intracellularly. Moreover, a bell-shaped correlation was observed between CA concentration and secretory EDIII titer. The maximum EDIII expression level of 187 mg/L was achieved under shake flask conditions with induction at 20 °C in the presence of 1 % CA. The overall increase in EDIII titer was ~9-fold compared to un-optimized conditions. Notably, mouse immune-sera, generated using this purified EDIII antigen, efficiently neutralized the DENV.ConclusionsThe strategy described herein could enable fulfilling the mounting demand for recombinant EDIII as well as lay direction to future studies on secretory expression of recombinant proteins in P. pastoris with CA as a media supplement.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0243-3) contains supplementary material, which is available to authorized users.
Highlights
Dengue is a viral disease spread to humans by mosquitoes
This study assesses the effect of low temperature induction and Casamino acids (CA) supplementation on the secretory titers of dengue virus (DENV)-3 Envelope domain-III (EDIII) in shake-flask settings
Optimization experiments were performed in minimal media instead of complex media owing to the ease in purification of secreted recombinant protein from minimal media
Summary
There are four serotypes of dengue viruses (DENV) that places ~40 % of the global population at risk of infection. Envelope domain-III (EDIII) antigen of dengue virus (DENV) has garnered much attention as a promising vaccine candidate for dengue, in addition to its use as a diagnostic intermediate. A mosquito-borne disease, can be caused by four antigenically distinct serotypes of dengue viruses (DENVs). Because of its unique features, recombinant versions of EDIII have found extensive utility in basic research, for understanding host-virus interactions, and immune response against natural infection [2, 3]. Numerous expression systems have been reported for the production of recombinant EDIII from different DENV serotypes, E. coli remains the most widely used expression host [4,5,6]. In the absence of a solubility tag, overexpressed EDIII tends to be insoluble and necessitates purification under denaturing conditions, followed by renaturation [4, 6]
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