METHODS: Optimization of culture medium for production of recombinant dengue protein in Escherichia coli

Enhanced production of recombinant dengue multi-epitope protein in Escherichia coli was achieved by optimization of culture medium. Complex media (Luria Bertani broth, Terrific broth, Super broth, M9 minimal media, five times Luria Bertani broth, semi-defined medium enriched with tryptone and yeast extract, and semi-defined medium enriched with glucose) were evaluated for production of recombinant dengue multi-epitope protein in shake flasks. The recombinant protein was further produced by fed-batch fermentation using 5 L bioreactor. Cells were grown in optimized semi-defined medium, and feeding was carried out with 5X medium and glycerol. When growth reached 14.35 g/L of dry cell weight, culture was induced with 0.5 mM IPTG and further grown for 4 h to reach 18.37 g/L dry cell weight. The recombinant dengue multi-epitope protein was purified from inclusion bodies under denaturing conditions using metal affinity chromatography, which yielded 96.43 mg/L of protein. The purified protein was found to be reactive with dengue-infected human serum samples. Introduction engue virus infection is recognized as a major mosquitoborne human infection of the 21st century. Recombinant dengue envelope (E) and nonstructural (NS1) protein have been well studied as antigens in diagnostic assays for detection of dengue infection1-3 and as reagents for vaccine studies.4-5 Escherichia coli is commonly used to produce recombinant proteins because it can be grown to high densities on inexpensive media and its genetics are well understood. Recombinant E. coli can be grown to high densities in common media such as Luria Bertani broth (LB),6-7 the synthetic M9 minimal salt medium, Terrific broth (TB),8-9 and Super broth (SB).10 The optimal growth temperature for E. coli is 37 °C, and pH between 6.4 and 7.2. The bacterium is generally grown under aerobic conditions since anaerobic growth provides less energy for metabolic processes such as protein synthesis.11 To ensure that oxygen supply does not become limiting, fedbatch operations are used extensively.6,12 This improves biomass and recombinant protein yield, over batch culture. Fed-batch operation overcomes possible limitations due to high concentrations of substrate and enables growth to be prolonged compared to traditional batch fermentations. High-cell-density fed-batch fermentation using glucose as the main carbon and energy source is a standard technique in E. coli cultivation.13,14 However, accumulation of acetate and other acidic byproducts during fed-batch cultivation diminishes cell growth as well Optimization of culture medium for production of recombinant dengue protein in Escherichia coli