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Casamino acids facilitate the secretion of recombinant dengue virus serotype-3 envelope domain III in Pichia pastoris
- Source :
- BMC Biotechnology
- Publication Year :
- 2015
-
Abstract
- Background Dengue 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. Results In 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. Conclusions The 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 material The online version of this article (doi:10.1186/s12896-016-0243-3) contains supplementary material, which is available to authorized users.
- Subjects :
- 0106 biological sciences
0301 basic medicine
Envelope domain III
Dengue virus
Biology
medicine.disease_cause
01 natural sciences
Pichia Pastoris
Pichia
Microbiology
Pichia pastoris
law.invention
Dengue fever
03 medical and health sciences
Mice
Antigen
Viral Envelope Proteins
law
010608 biotechnology
medicine
Animals
Secretion
Amino Acids
Mice, Inbred BALB C
Dengue Virus
biology.organism_classification
medicine.disease
Casamino acids
Virology
Recombinant Proteins
Culture Media
Titer
030104 developmental biology
Recombinant DNA
Biotechnology
Research Article
Subjects
Details
- ISSN :
- 14726750
- Volume :
- 16
- Database :
- OpenAIRE
- Journal :
- BMC biotechnology
- Accession number :
- edsair.doi.dedup.....4d3741e1a4a68004376532803c15ada5