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Microbiology and Biotechnology Letters


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Microbial Biotechnology (MB)  |  Synthetic Biology and Metabolic Engineering

Microbiol. Biotechnol. Lett. 2021; 49(2): 148-156

Received: June 9, 2020; Accepted: January 26, 2021

The Effect of Growth Condition on a Soluble Expression of Anti-EGFRvIII Single-chain Antibody in Escherichia coli NiCo21(DE3)

Kartika Sari Dewi1*, Ratna Annisa Utami2, Hariyatun Hariyatun1, Riyona Desvy Pratiwi , Dian Fitria Agustiyanti1, and Asrul Muhamad Fuad1

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

Correspondence to :
Kartika Sari Dewi,

Single-chain antibodies against epidermal growth factor receptor variant III (EGFRvIII) are potentially promising agents for developing antibody-based cancer treatment strategies. We described in our previous study the successful expression of an anti-EGFRvIII scFv antibody in Escherichia coli. However, we could also observe the formation of insoluble aggregates in the periplasmic space, limiting the production yield of the active product. In the present study, we investigated the mechanisms by which growth conditions could affect the expression of the soluble anti-EGFRvIII scFv antibody in small-scale E. coli NiCo21(DE3) cultures, attempting to maximize production. The secreted scFv molecules were purified using Ni-NTA magnetic beads and protein characterization was performed using SDS-PAGE and western blot analyses. We used the ImageJ software for protein quantification and determined the antigen-binding activity of the scFv antibody against the EGFRvIII protein. Our results showed that the highest percentage of soluble scFv expression could be achieved under culture conditions that combined low IPTG concentration (0.1 mM), low growth temperature (18℃), and large culture dish surface area. We found moderate-yield soluble scFv production in the culture medium after lactose-mediated induction, which was also beneficial for downstream protein processing. These findings were confirmed by conducting western blot analysis, indicating that the soluble, approximately 30-kDa scFv molecule was localized in the periplasm and the extracellular space. Moreover, the antigen-binding assay confirmed the scFv affinity against the EGFRvIII antigen. In conclusion, our study reveals that low-speed protein expression is preferable to obtain more soluble anti-EGFRvIII scFv protein in an E. coli expression system.

Keywords: Escherichia coli, EGFRvIII, scFv, periplasmic expression, PelB signal peptide

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