Specific N-Terminal amino acids potentiate the periplasmic expression of single-chain variable fragments in Escherichia coli

Abstract

Single-chain variable fragments (ScFvs) are important in therapy, diagnosis and research because of their elevated antigen affinity and low immunogenicity. At present, high-yield scFv expression in Escherichia coli is limited by insoluble aggregation in the reducing environment of the cytoplasm or low yields in the periplasm. Here we achieved increased expression of scFvs in the periplasm by inserting optimal amino acids between the signal peptide and scFv. We constructed an expression library with three random amino acids at the scFv N-Terminus, screened this library with a single-step colony assay and identified the specific sequences that boosted periplasmic expression of scFvs. METHOD SUMMARY A library of single-chain variable fragments with three random amino acids at the N-Terminus was constructed. The library was screened using a single-step colony assay. This simple and rapid screening method enabled the identification of specific sequences responsible for improved periplasmic expression, without false-positive clones.