Journal
FRONTIERS IN MOLECULAR BIOSCIENCES
Volume 9, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmolb.2022.848829
Keywords
signal peptide; inducer; Escherichia coli; anti-GFP VHHs; inclusion body; soluble protein
Categories
Funding
- National Natural Science Foundation of China
- Natural Science Foundation of Liaoning Province in China [32070936, 2021JH6/10500147]
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In this study, the expression system of anti-GFP VHHs in E. coli was optimized, and it was found that signal peptides like OmpA and PelB can efficiently enhance the solubility of VHHs without affecting their bioactivity. This provides a novel strategy for the industrial production of soluble VHHs and the research of other VHHs.
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 beta-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.
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