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. 2025 Jul 3.
doi: 10.1007/s12033-025-01468-8. Online ahead of print.

Extracellular Expression and Diagnostic Potential of Dengue Virus Type 3 E Protein Domain III Through Codon Optimization in Komagataella phaffii

Camila Marques de Simone  1 , Maria Carolina Yi Lin Lee  1 , Yasmin Mayra Bispo  1 , Marcos Benedito Adão  1 , Leonardo de Paiva Turetta  1 , Marita Vedovelli Cardozo  1 , Monalessa Fábia Pereira  1 , Christiane Eliza Motta Duarte  2 , Sérgio Oliveira De Paula  3 , Caio Roberto Soares Bragança  4
Affiliations

Extracellular Expression and Diagnostic Potential of Dengue Virus Type 3 E Protein Domain III Through Codon Optimization in Komagataella phaffii

Camila Marques de Simone et al. Mol Biotechnol. .

Abstract

Dengue is a mosquito-borne viral disease caused by the dengue virus (DENV), which affects millions worldwide. The envelope (E) protein, particularly domain III (EDIII), plays a critical role in viral entry and is a major target for diagnostic and vaccine development. In this study, we aimed to express and purify EDIII from DENV-3 using Komagataella phaffii as an expression system. The recombinant gene was codonoptimized, cloned into the pPICZαA vector, and integrated into the K. phaffii genome. Protein expression was induced with methanol and purified using Ni2+-affinity chromatography, yielding 209-309 mg/L. SDS-PAGE and Western blot confirmed protein expression. The recombinant antigen was evaluated in an indirect ELISA with 92 serum samples, demonstrating an AUC of 0.94 (IgM) and 0.828 (IgG), with 92% specificity for IgM. These results suggest that K. phaffii is a suitable host for recombinant dengue antigen production, offering a scalable and cost-effective strategy for diagnostic applications.

Keywords: Bioprocess; Codon optimization; DENV; Dengue; Domain III (EDIII); Heterologous expression; Recombinant protein; Yeast.

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