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

Abstract

scFv-BM3 is a single-chain variable fragment (scFv) against aflatoxin B₁ (AFB₁ ) 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 AFB₁ , 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 AFB₁ . 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 β-sheet structure and adopted a proper conformation to bind AFB₁ with high affinity and specificity in various biophysical and biochemical analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: Single-chain variable fragments (scFvs) are recombinant antibodies that are difficult to produce as a functional form in Escherichia coli. This study demonstrates the production of functional scFvs against aflatoxin B₁ (AFB₁ ) (scFv-BM3) using Escherichia coli by extracellular secretion. While periplasmic expression of scFv-BM3 resulted in formation of inactive aggregates in E. coli, the scFv-BM3 secreted into the culture medium adopted a properly folded structure for specific binding to AFB₁ . This study promotes the application of functional scFv-BM3 to the immunological detection of AFB₁ in biotechnology fields.

Keywords: Escherichia coli; Triton X-100; aflatoxin B1; extracellular production; single-chain variable fragment.