Annales Bogorienses Vol. 21, No. 1, 2017 29 Comparison of Gene Expression between Two Types of Anti-EGFRvIII ScFv Antibodies Having Different Variable Domain Orders In Escherichia coli Kartika Sari Dewi* and Asrul Muhamad Fuad Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Jalan Raya Bogor Km 46, 16911, Cibinong, Bogor, Indonesia. Abstract Several studies reported that the expression of various kinds of single-chain variable fragment (scFv) antibodies in Escherichia coli are significantly influenced by the order of their variable domains. To date, the effect of the order of variable domains in the expression of scFv antibodies against epidermal growth factor receptor variant III (EGFRvIII) has not been reported. This study aimed to compare the expression between VH- linker-VL and VL-linker-VH domain orders of the anti-EGFRvIII scFv antibodies in E. coli expression system. Recombinant plasmids inserted with DNA encoding scFv proteins were transformed into E. coli NiCo21 (DE3) competent cells and characterized by colony PCR. The expression of scFv proteins was done by using optimum concentration of inducer. Total proteins, soluble periplasmic and cytoplasmic proteins, also extracellular proteins were isolated, subsequently characterized by SDS-PAGE, Slot Blot, and ImageJ software analyses. The antigen- binding activity of both scFvs proteins against EGFRvIII was observed. The results showed that the relative percentage of scFv expression with VH-linker-VL domain order is higher than that of VL-linker-VH in each compartment. Moreover, both of scFvs proteins have antigen-binding activity against EGFRvIII. Keywords: EGFRvIII, scFv, variable domain order, antibody, Escherichia coli ---------------------------- * Corresponding author: Cibinong Science Center, Jl. Raya Bogor Km. 46, Cibinong 16911, Indonesia Tel. +62-21-8754587, Fax. +62-21-87754588 E-mail: kart008@lipi.go.id Introduction The type III epidermal growth factor receptor (EGFRvIII) is a mutant variant of EGFR that has in-frame deletion of 2-7 exons in mRNA, resulting in the deletion of amino acids 6-273 in the extracellular domain and the formation of a new Glycine in the junction of exon 1 and 8 (Wikstrand et al., 1998). EGFRvIII is not found in normal cells, but is the most common mutation observed in a number of human carcinomas, including gliomas, ovarian carcinomas, non-small-cell lung carcinomas, prostate carcinomas, and breast carcinomas (Gan et al., 2013). This biological characteristic of EGFRvIII makes it a highly promising therapeutic target for immunology-based cancer therapy (Greenall & Johns, 2016). Furthermore, single-chain variable fragment (scFv) antibodies are powerful tools for such targeted therapy. ScFv is a minimized antibody consisting of heavy-chain (VH) and light-chain (VL) variable domains that are connected by a short peptide linker to stabilize the molecule. It has better pharmacokinetic properties comparing to its parental monoclonal antibody including better tumor penetration, lower retention time in non-target tissue, and lower immunogenicity. ScFv represents the smallest functional VH-VL of an antibody which is necessary for high-affinity binding of antigen. Because of its small size (~30 kDa), scFv can be easily expressed in a functional form in the bacterial system, offering the protein engineering to improve their properties such as increase of antigen-binding activity and specificity (Ahmad et al., 2012). However, scFv is known to have two disulfide bonds which contribute to their antigen-binding activity and stability. Good expression levels for scFv in E. coli expression system can be achieved via soluble