Extracellular production of functional single-chain variable fragment against aflatoxin B1 using Escherichia coli

scFv-BM3 is a single-chain variable fragment (scFv) against aflatoxin B-1 (AFB(1)) engineered by affinity maturation and site-directed mutagenesis, and thus has a 31-fold higher affinity than its wild-type. To apply scFv-BM3 to immunological detection of AFB(1), periplasmic expression in Escherichia coli was attempted to produce a functional form of scFv-BM3. scFv-BM3 accumulated as inactive aggregates in the cells. However, it was found that scFv-BM3 secreted into the culture medium had binding activity to AFB(1). Expression conditions for scFv-BM3 were further manipulated to enhance secretion into the culture medium. This extracellular secretion of functional scFv-BM3 was significantly improved by supplementation with Triton X-100 and optimization of expression conditions. The scFv-BM3 purified from the culture medium exhibited a typical antiparallel beta-sheet structure and adopted a proper conformation to bind AFB(1) with high affinity and specificity in various biophysical and biochemical analyses.