Expression of scFv-anti-CHIKV-E2 in Escherichia coli with chaperones co-expression, and its functional assay by electrochemical immunosensor

• Published in: Journal of immunoassay & immunochemistry Vol. 45; no. 4; pp. 307 - 324
• Main Authors: Gaffar, Shabarni, Nurbayanti, Siti Hesti, Hartati, Yeni Wahyuni, Novianti, Mia Tria, Novitriani, Korry, Ishmayana, Safri, Yusuf, Muhammad, Subroto, Toto
• Format: Journal Article
• Language: English
• Published: New York Taylor & Francis 03-07-2024; Marcel Dekker, Inc
• Subjects: Chaperones; chaperones - co-expression; E coli; electrochemical immunosensor; Electrochemistry; Escherichia coli; gold modified SPCE; Immunoassay; Immunosensors; Inclusion bodies; Monoclonal antibodies; Potassium ferricyanide; Protein folding; scFv-anti-CHIKV-E2; Trigger factor
• ISSN: 1532-1819; 1532-4230; 1532-4230
• DOI: 10.1080/15321819.2024.2356639

Single Chain Variable Fragment (scFv), a small fragment of antibody can be used to substitute the monoclonal antibody for diagnostic purposes. Production of scFv in Escherichia coli host has been a challenge due to the potential miss-folding and formation of inclusion bodies. This study aimed to express scFv-anti-CHIKV-E2 which previously designed specifically for Asian strains by co-expression of three chaperones that play a role in increasing protein solubility; GroEL, GroES, and Trigger Factor. The scFv and chaperones were expressed in Origami B E. coli host under the control of the T7 promoter, and purified using a Ni-NTA column. Functional assay of scFv-anti-CHIKV-E2 was examined by electrochemical immunosensor using gold modified Screen Printed Carbon Electrode (SPCE), and characterized by differential pulses voltammetry (DPV) using K
3
[Fe(CN)
6
] redox system and scanning microscope electron (SEM). The experimental condition was optimized using the Box-Behnken design. The results showed that co-expression of chaperone increased the soluble scFv yield from 54.405 μg/mL to 220.097 µg/mL (~5×). Furthermore, scFv can be used to detect CHIKV-E2 in immunosensor electrochemistry with a detection limit of 0.74048 ng/mL and a quantification limit of 2.24388  ng/mL. Thus, the scFv-anti-CHIKV-E2 can be applied as a bioreceptor in another immunoassay method.