Production of functional single-chain variable fragment specific for food-born mycotoxin, aflatoxin B1 in engineered Escherichia coli

Abstract

Aflatoxin B1 (AFB1) is a secondary fungal metabolite produced by Aspergillus flavus and A. parasiticus. International Agency of Research on Cancer (IARC) classified AFB1 into a group I carcinogen for humans. Aflatoxin B1 contaminate grains and crops that human and livestock consume. Detection of aflatoxin B1 is an essential step to reduce this threat. Three mtehods have been used to detect aflatoxin B1

biological, analytical, and immunological methods. Enzyme immunosorbent assay (ELISA) is highly specific, sensitive, simple and rapid for measuring aflatoxins in foods. In previous research, scFv was cloned from the murine monoclonal antibody to aflatoxin B1 and produced in E. coli. However, the scFv was expressed in insoluble form, so an in vitro refolding procedure was necessary to acquire soluble and active scFv. In the following previous research, by co-expressing several folding modulators from E. coli and Bacillus subtilis with scFv in E coli, soluble scFv was produced and secreted into the periplasm of E. coli. Even though soluble scFv was produced, the proportion of the soluble scFv in the total expression level was low. Thus, a new expression system needs to be developed to produce soluble scFv in large proportion. To increase the proportion of soluble scFv, maltose binding protein (MBP) fusion, codon optimization, and new E. coli C41(DE3) strain used as a host were introduced. First, MBP fusion with the scFv gene for enhancing an expression level in soluble form of scFv was attempted. A MBP was fused with the N or C-terminal of scFv. An expression level of the fused scFv slightly increased due to the MBP fusion. An expression level of the C-terminal MBP fused scFv was higher than N-terminal fusion. However, the expression form of scFv was still insoluble. Codon optimization of the scFv gene was adopted. Elimination of codon usage bias makes the heterologous protein expression easier. The codon optimized scFv fused with MBP were expressed in E. coli BL21(DE3). Despite codon optimization, it was not effective to produce in soluble form. Thirdly, besides BL21(DE3), other E. coli host strains were selected to express scFv. C41(DE3) and Origami (DE3) strains were employed to express the MBP-fused scFv. The C41(DE3) strain carrying the scFv fused N-terminal MBP was able to produce soluble and active scFv with the high proportion of the total expression scFv. A fed-batch fermentation was conducted to maximize the production of the scFv-MBP. The maximum concentration of scFv was 810 mg/L. The scFv-MBP fusion protein was purified with affinity chromatography using hisitidine tags for characterizing the antibody properties. The produced scFv was tested for antigen-binding activity by indirect ELISA. The antigen-binding activity was determined by the absorbance of the MBP-fused scFv being proportional to concentrations aflatoxin B1. The secondary structure of the scFv protein was analyzed by circular dichroism in range of 190 ~ 250 nm. The beta sheet structure was identified as the secondary structure of scFv. The new expression system consisting of the C41(DE3) strain carrying the scFv fused N-terminal MBP with the signal sequence of MBP was applied to other types of scFv such as fumonisin B1 and deoxynivalenol. The two scFv of fumonisin B1 and deoxynivalenol were successfully expressed in soluble form. In conclusion, this thesis allowed the development of a novel expression system for soluble production of various scFv in E. coli.