Background Overexpression of the EpCAM (epithelial cell adhesion molecule) in malignancies makes it an attractive target for passive immunotherapy in a wide range of carcinomas. In comparison with full-length antibodies, due to the small size, the scFvs (single-chain variable fragments) are more suitable for recombinant expression in E. coli (Escherichia coli). However, the proteins expressed in large amounts in E. coli tend to form inclusion bodies that need to be refolded which may result in poor recovery of bioactive proteins. Various engineered strains were shown to be able to alleviate the insolubility problem. Here, we studied the impact of four E. coli strains on the soluble level of anti-EpEX-scFv (anti-EpCAM extracellular domain-scFv) protein. Results Although results showed that the amount of soluble anti-EpEX-scFv obtained in BL21TM (DE3) (114.22 ± 3.47 mg/L) was significantly higher to those produced in the same condition in E. coli RosettaTM (DE3) (71.39 ± 0.31 mg/L), and OrigamiTM T7 (58.99 ± 0.44 mg/L) strains, it was not significantly different from that produced by E. coli SHuffleTM T7 (108.87 ± 2.71 mg/L). Furthermore, the highest volumetric productivity of protein reached 318.29 ± 26.38 mg/L in BL21TM (DE3). Conclusions Although BL21TM (DE3) can be a suitable strain for high-level production of anti-EpEX-scFv protein, due to higher solubility yield (about 55%), E. coli SHuffleTM T7 seems to be better candidate for soluble production of scfv compared to BL21TM (DE3) (solubility yield of about 30%).

Background and purpose: The epithelial cell adhesion molecule (EpCAM), is one of the first cancer- associated markers discovered. Its overexpression in cancer stem cells, epithelial tumors, and circulating tumor cells makes this molecule interesting for targeted cancer therapy. So, in recent years scFv fragments have been developed for EpCAM targeting. Experimental approach: In this study, an scFv against EpCAM extracellular domain (EpEX) derived from 4D5MOC-B humanized mAb was expressed in Escherichia coli k12 strain, and in order to obtain the optimum culture conditions in chemically defined minimal medium, response surface methodology (RSM) was employed. According to the RSM-CCD method, a total of 30 experiments were designed to investigate the effects of various parameters including isopropyl-b-D-thiogalactopyranoside (IPTG) concentration, cell density before induction, post-induction time, and post-induction temperature on anti EpEX-scFv expression level. Findings/Results: At the optimum conditions (induction at cell density 0.8 with 0.8 mM IPTG for 24 h at 37 °C), the recombinant anti EpEX-scFv was produced at a titer of 197.33 μg/mL that was significantly consistent with the prediction of the model. Conclusion and implication: The optimized-culture conditions obtained here for efficient production of anti EpEX-scFv in shake flask cultivation on a chemically defined minimal medium could be applied to large- scale fermentation for the anti EpEX-scFv production.

Escherichia coli is an attractive and cost-effective cell factory for producing recombinant proteins such as single-chain variable fragments (scFvs). AntiEpEX-scFv is a small antibody fragment that has received considerable attention for its ability to target the epithelial cell adhesion molecule (EpCAM), a cancerassociated biomarker of solid tumors. Due to its metabolic burden, scFv recombinant expression causes a remarkable decrease in the maximum speci c growth rate of the scFv-producing strain. In the present study, a genome-scale metabolic model (GEM)-guided engineering strategy is proposed to identify gene targets for improved antiEpEX-scFv production in E. coli. For improving scFv production, ux balance analysis (FBA) and FVSEOF methods identi ed a number of potential genetic engineering targets localized in the glucose uptake system and pentose phosphate pathway. From the predicted targets, glk gene encoding glucokinase was chosen to be overexpressed in the parent strain Escherichia coli BW25113 (DE3). By overexpressing glk, the growth capacity of the recombinant E. coli strain was recovered. Moreover, the engineered strain with glk overexpression successfully lead to increased scFv production. This approach can be considered for the improvement of other recombinant proteins production.

Increasing demand for recombinant therapeutic proteins highlights the necessity of their yield improvement. Culture medium formulation is a popular approach for bioprocess optimization to improve therapeutic protein production. Constraint-based modeling can empower high-precision optimization through information on how media compounds affect metabolism and cell growth. In the current study, a genome-scale metabolic model (GEMM) of Escherichia coli cells was employed to design strategies of minimal medium supplementation for higher antiEpEX-scFv production. Dynamic flux balance analysis of the recombinant E. coli cell model predicted that ammonium was depleted during the process. Based on the simulations, three amino acids (Asn, Gln and Arg) were chosen to be added to the medium to compensate for ammonium depletion. Experimental validation suggested that the addition of these amino acids (one-by-one, or in combinations) can indeed improve cell growth and recombinant protein production. Then, design of experiment was used to optimize the concentrations of amino acids in the growth medium. About two-fold increase in the growth rate and total scFv expression level was observed using this strategy. We conclude that the GEMM-based approach can provide insights into an effective feeding strategy to improve the production of recombinant protein in E. coli.

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