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

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.

Extracellular Expression of Feruloyl Esterase and Xylanase in Escherichia coli for Ferulic Acid Production from Agricultural Residues

“…It is generally considered that the signal peptide selection is a simple and effective strategy for recombinant protein secretion, aiding to promote its soluble expression, reduce inclusion body formation, improve enzyme activity, and facilitate downstream processing [ 23 , 24 , 25 ]. In order to realize the secretory expressions of FAE, the fae gene was ligated into the pET-22b(+) vector to be fused with the intrinsic signal peptide pelB.…”

“…In order to optimize the secretory expression of FAE and XYN, five levels of initial cell density (OD 600 = 0.5, 0.8, 1.0, 1.5, and 2.0, respectively), five levels of IPTG concentration (0.05, 0.1, 0.2, 0.5, and 1.0 mM, respectively), five levels of induction temperature (16,20,25,30, and 37 • C, respectively), as well as three levels of glycine concentration (2, 5, and 10 g/L, respectively) were all evaluated while maintaining the other parameters at a constant level. Experiments were performed in a 250 mL flask containing 50 mL of the medium.…”

“…For XYN, its extracellular activity accounted for 51.6% of the total after optimization, which was only 24.5% before optimization, respectively (Figure 4B). Single-factor analysis on FAE and XYN secretion with (A) five levels of initial cell density (OD 600 = 0.5, 0.8, 1.0, 1.5, and 2.0, respectively), (B) five levels of IPTG concentration (0.05, 0.1, 0.2, 0.5, and 1.0 mM, respectively), (C) five levels of induction temperature (16,20,25,30, and 37 • C, respectively), and (D) three levels of glycine concentration (2, 5, and 10 g/L, respectively). Data reflect the mean ± SD (n = 3).…”

Scalable, Robust, High-throughput Expression, Purification & Characterization of Nanobodies Enabled by 2-Stage Dynamic Control

“…In E. coli , periplasmic expression of VHHs typically yields titers in the range of tens of milligrams per liter, whereas cytoplasmic expression has achieved up to ∼200 mg/L in both SHuffle and BL21(DE3) strains. 15,24 Beyond intracellular processes, extracellular expression (via protein export) has resulted in more impressive yields with titers peaking at 400 mg/L 25 . However, this approach requires optimization of various factors including the signal peptide used for export, strain, and induction method for every protein target.…”

A selection and optimization strategy for single-domain antibodies targeting the PHF6 linear peptide within the tau intrinsically disordered protein

“…They present several advantages such as an easy recombinant production in different systems be it prokaryotes, yeasts, or insect cells and more ( 31 ), good stability and engineering properties ( 15 ). This leads to many opportunities to optimize those VHHs for specific use, considering their production ( 32 ), their affinity ( 33 ), their stability ( 34 ), or their capacity of being produced intracellularly ( 9 , 16 , 35 ). In relation to this last point, a significant part of VHHs may be unable to fold properly in the reducing environment of the cytoplasm ( 4 , 36 ), which inhibits the formation of the disulfide bridges that contribute to the VHH stability, leading to aggregation or degradation ( 35 , 37 ).…”