Highly Expressed Soluble Recombinant Anti-GFP VHHs in Escherichia coli via Optimized Signal Peptides, Strains, and Inducers

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 stud...

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Published inFrontiers in molecular biosciences Vol. 9; p. 848829
Main Authors Chao, Shuangying, Liu, Yuhang, Ding, Ning, Lin, Yue, Wang, Qian, Tan, Junwen, Li, Wei, Zheng, Yang, Hu, Xuejun, Li, Junming
Format Journal Article
LanguageEnglish
Published Switzerland Frontiers Media S.A 10.03.2022
Subjects
anti-GFP VHHs
Escherichia coli
inclusion body
inducer
Molecular Biosciences
signal peptide
soluble protein
inclusion body
signal peptide
inducer
soluble protein
Escherichia coli
anti-GFP VHHs
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Summary: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 strains 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) 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 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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Reviewed by: Samanta Raboni, University of Parma, Italy
These authors jointly supervised this work
Edited by: Andrea Mozzarelli, University of Parma, Italy
These authors have contributed equally to this work
This article was submitted to Protein Biochemistry for Basic and Applied Sciences, a section of the journal Frontiers in Molecular Biosciences
Mehmet Berkmen, New England Biolabs, United States
ISSN:2296-889X
2296-889X
DOI:10.3389/fmolb.2022.848829