Abstract
The relationship between the expression of HER2 and malignity of breast tumors has led to the generation of antibodies targeting HER2+ tumors. In addition, the expression of scFvs, as the smallest antigen-binding region of antibody containing two disulfide bonds in Escherichia coli often results in accumulating non-functional protein in the cytoplasm. A redox-modified strain of E. coli such as Origami (DE3) may facilitate the formation of proper disulfide bond in cytoplasm. The present study aimed to optimize the expression of anti-HER2 scFv in Origami and evaluate the influence of induction temperature, and host strain on the solubility of the protein. To this aim, chemicallysynthesized anti-HER2 scFv of Trastuzumab was cloned in pET-22b (+). The results demonstrated that anti-HER2 scFv is expressed in Origami, purified by using Ni-NTA column, and detected by anti-His antibody in Western blot analysis. The highest anti-HER2 scFv expression in Origami was achieved 24 h after IPTG induction (1 mM) at 37 oC. Further, the total anti-HER2 scFv expression level was higher in BL21, compared to Origami strain. However, the ratio of soluble/insoluble forms of anti-HER2 scFv increased in Origami strain. Furthermore, higher soluble expression was achieved when the culture of recombinant Origami was conducted at lower temperature (25 oC).
Highlights
The overexpression of HER2 receptor has been usually observed in 20-30 % of breast cancers (Kiewe et al, 2006)
Single chain fragment variable, as the most widely used antibody fragment, includes the variable heavy (VH) and variable light (VL) domains of an antibody which linked together by a flexible polypeptide linker (Ahmad et al, 2012). scFv antibody demonstrates faster pharmacokinetics and potentially more homogenous tumor penetration related to large IgG molecules due to the reduced size and lack of Fc domain (Kelly et al, 2008). scFvs have been used in different therapeutic such as immunoliposomes (Park et al, 2002), immunotoxins and radioimmunotherapy (Cao et al, 2012; Cao et al, 2009; Kelly et al, 2008), as well as diagnostic strategies like radioimmunodetection (Kelly et al, 2008; Pucca et al, 2011)
The expression of the recombinant anti-HER2 scFv in Origami (DE3) host was induced by 1 mM IPTG at 37 °C
Summary
The overexpression of HER2 receptor has been usually observed in 20-30 % of breast cancers (Kiewe et al, 2006). The overexpression of scFv proteins with two disulfide-bond in E. coli cytoplasm results in losing correct conformation and accumulation into insoluble aggregates and non-functional proteins (Guglielmi, Martineau, 2009) In this regard, many strategies consider the expression of proteins containing disulfide bonds in the E. coli cytoplasm because of these limitations. The trxB and gor negative strains of E. coli alone or in combination with the expression of foldases/chaperones are commonly accepted and effectively exploited (De Marco, 2009) Among these strains, Origami from Novagen with mutation in trxB and gor genes can improve the folding of recombinant proteins containing disulfide bond in the E. coli cytoplasm (Salinas et al, 2011; Sørensen, Mortensen, 2005). The influence of isopropyl-beta-Dthiogalactopyranoside (IPTG) concentrations, duration and temperature of induction, as well as strain type on the expression of anti-HER2 scFv was evaluated in this study
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