Abstract
Antigen-binding variable domains of the H chain of heavy-chain antibodies (VHHs), also known as nanobodies (Nbs), are of great interest in imaging technique, disease prevention, diagnosis, and therapy. High-level expression of soluble Nbs is very important for its industrial production. In this study, we optimized the expression system of anti-green fluorescent protein (GFP) VHHs with three different signal peptides (SPs), outer-membrane protein A (OmpA), pectate lyase B (PelB), and L-asparaginase II SP (L-AsPsII), in different Escherichia coli strains via isopropyl β-D-thiogalactoside (IPTG) induction and auto-induction, respectively. The solubility of recombinant anti-GFP VHHs with PelB or OmpA was significantly enhanced to the same extent by IPTG induction and auto-induction in BL21 (DE3) E. coli strain and the maximum yield of target protein reached approximately 0.4 mg/l in a shake flask. The binding activity of recombinant anti-GFP VHHs was also confirmed to be retained by native-polyacrylamide gel electrophoresis (PAGE). These results suggest that SPs like OmpA and PelB could efficiently improve the recombinant anti-GFP VHH solubility without changing its bioactivity, providing a novel strategy to optimize the E. coli expression system of soluble VHHs, and lay the foundation for the industrial production of soluble recombinant anti-GFP VHHs and the research of other VHHs in the future.
Highlights
Recombinant protein production via prokaryotic system has brought hundreds of therapeutic proteins into clinical applications or clinical trials over the past few decades (Dimitrov, 2012; Sánchez-Trasviña et al, 2021)
Since it was reported that several anti-green fluorescent protein (GFP) VHH mutants were found to possess enhanced binding activities by changing their amino acid sequences of the complementarity determining region 3 (CDR3) via phage display technique (Fang et al, 2020), we used the best-performing mutants as the model recombinant VHHs in this work
Each plasmid was transformed into BL21 (DE3), and the protein expression was induced by isopropyl β-D-thiogalactoside (IPTG)
Summary
Recombinant protein production via prokaryotic system has brought hundreds of therapeutic proteins into clinical applications or clinical trials over the past few decades (Dimitrov, 2012; Sánchez-Trasviña et al, 2021). In some cases, when high-level expression of recombinant proteins in Escherichia coli occurs, insoluble aggregates accumulate as inclusion bodies. Numerous strategies were attempted to overcome this problem, such as the optimization of bacterial strains, promoters, inducers, as well as the use of N-terminal signal peptides (SPs), which could help recombinant proteins be secreted into the culture medium (Tegel et al, 2011; Chen, 2012; Freudl, 2018; Owji et al, 2018; Liu et al, 2019). Extracellular production of proteins possesses several advantages. Extracellular expression could prevent aggregations of proteins and accumulation of inclusion
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