Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner.

Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner. Appl Microbiol Biotechnol. 2019 Jun 07;: Authors: Liu M, Wang B, Wang F, Yang Z, Gao D, Zhang C, Ma L, Yu X Abstract Single-chain variable fragment (scFv) has great prospect in medical therapies and diagnostic applications due to its binding affinity and low immunogenicity. However, the application of scFv is limited by its heterologous expression facing challenges of insoluble aggregation. sfGFP has been developed as fusion tag to facilitate the solubility of fusion partner in Escherichia coli. We designed fusion protein of anti-influenza PB2 scFv at C-terminus of sfGFP and successfully obtained soluble expression of sfGFP-scFv-His in Escherichia coli. The expression level of sfGFP-scFv-His reached at 20 mg/L of bacterial culture when the culture was induced with 0.1 mM IPTG at 18 °C for 16 h. And 6 mg scFv-His was obtained from the cleavage of 10 mg pure sfGFP-scFv-His with TEV protease. In addition, we found that sfGFP-scFv-His was more stable than scFv-His in chicken serum, suggesting that sfGFP not only facilitated the solubility of scFv in Escherichia coli, but also promoted the stability of scFv. The immunologic activity of sfGFP-scFv-His was confirmed by Western blot and ELISA; the results showed that anti-PB2 sfGFP-scFv-His exhibited specific binding to PB2. Hemagglutination an...
Source: Applied Microbiology and Biotechnology - Category: Microbiology Authors: Tags: Appl Microbiol Biotechnol Source Type: research