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Microbial Biotechnology | Synthetic Biology and Metabolic Engineering
Microbiol. Biotechnol. Lett.
Kartika Sari Dewi 1*, Ratna Annisa Utami 2, Hariyatun Hariyatun 1, Riyona Desvy Pratiwi 1, Dian Fitria Agustiyanti 1 and Asrul Muhamad Fuad 1
1Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Jalan Raya Bogor Km. 46, Bogor 16911, Indonesia, 2School of Pharmacy, Institut Teknologi Bandung, Jalan Ganesha No. 10, Bandung 40132, Indonesia
Single-chain antibody against epidermal growth factor receptor variant III is a promising agent for antibody-based cancer treatment. Our previous study has successfully expressed anti-EGFRvIII scFv antibody in E. coli. However, its production was limited by the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. The present study investigated the effect of growth condition on the expression of soluble anti-EGFRvIII scFv antibody in small-scale E. coli NiCo21(DE3) cultures in an attempt to maximize production. Secreted scFv was purified using Ni-NTA magnetic beads and protein characterization was performed using SDS-PAGE and Western blot analyses. ImageJ software was used for protein quantification and the antigen-binding activity of scFv antibody was tested against EGFRvIII protein. The result showed that the highest percentage of soluble scFv expression was achieved when a culture was grown on the combination of low concentration of IPTG (0.1 mM), lower growth temperature (18°C), and larger surface area of a vessel. We found a moderate-yield of soluble scFv in culture medium under the lactose induction which also has a benefit for downstream protein processing. These findings were confirmed by Western blot analysis which showed that the soluble scFv can be localized in the periplasm and found in extracellular space with a size of around 30 kDa. Moreover, the antigen-binding assay exhibited the affinity of scFv against the EGFRvIII antigen. In conclusion, our study highlighted that the slower protein expression is preferred to get more soluble anti-EGFRvIII scFv protein in E. coli expression system.
Keywords: Escherichia coli, EGFRvIII, scFv, periplasmic expression, PelB signal peptide
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