Response surface methodology for optimization of scFv-BAD anti-E2 CHIKV expression in Escherichia coli Origami B (DE3) for the detection of Chikungunya virus

Shabarni Gaffar, Rachelia Salsabila Zulfa, Fadhil Adikoesoemo, Syifa Tazkiya, Korry Novitriani, Dani Permana, Sri Agung Fitri Kusuma, Hesti Lina Wiraswati, Mia Tria Novianti, Safri Ishmayana, Muhammad Yusuf, Toto Subroto
https://doi.org/10.1080/10826068.2025.2512944

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for recurring outbreaks, highlighting the need for rapid and accurate diagnostic tools. The E2 glycoprotein of CHIKV is a promising target for antibody-based detection. This study aims to determine the optimal expression conditions of recombinant single-chain variable fragment (scFv) fused with biotin acceptor domain (BAD) specific to the CHIKV E2 glycoprotein in Escherichia coli Origami B (DE3). The optimization was performed using the Box-Behnken design of response surface methodology (RSM), with the IPTG inducer concentration, induction time, and induction temperature as the independent variables. The optimal conditions were identified as 0.2 mM IPTG, 2 hours induction at 37 °C, resulting in a total protein concentration of 0.658 mg/mL. The soluble fraction of scFv-BAD was successfully purified using Ni²⁺-NTA affinity chromatography, with a purity of 91.11%. ELISA confirmed that the recombinant scFv-BAD was biotinylated and retained its ability to bind the CHIKV E2 antigen. The optimized scFv-BAD construct demonstrates potential for use in various immunoassay platforms, including rapid diagnostic tests for CHIKV detection.

Keywords:

Box-Behnken design, E. coli Origami B (DE3), expression optimization, scFv-BAD-anti-E2 CHIKV