title: Casamino acids facilitate the secretion of recombinant dengue virus serotype-3 envelope domain III in Pichia pastoris.
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.
authors: Centre for Biodesign and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India. nehakaushik@thsti.res.in.
authors: Neha
authors: N
authors: Kaushik
authors: Centre for Biodesign and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India. deepak.rohila@thsti.res.in.
authors: Deepak
authors: D
authors: Rohila
authors: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India. upasanaaro@gmail.com.
authors: Upasana
authors: U
authors: Arora
authors: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India. rajeraut@gmail.com.
authors: Rajendra
authors: R
authors: Raut
authors: Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland. urplammi@utu.fi.
authors: Urpo
authors: U
authors: Lamminmäki
authors: Recombinant Gene Products Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India. navinkhanna5@gmail.com.
authors: Navin
authors: N
authors: Khanna
authors: Centre for Biodesign and Diagnostics, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India. gaurav.batra@thsti.res.in.
authors: Gaurav
authors: G
authors: Batra
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D003470:Culture Media / Q000737:chemistry / Q000378:metabolism; D003716:Dengue Virus / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D003470:Culture Media; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C017721:casamino acids