Anti-EGFR VHH Antibody under Thermal Stress Is Better Solubilized with a Lysine than with an Arginine SEP Tag

Abstract

In this study, we assessed the potential of arginine and lysine solubility-enhancing peptide (SEP) tags to control the solubility of a model protein, anti-EGFR VHH-7D12, in a thermally denatured state at a high temperature. We produced VHH-7D12 antibodies attached with a C-terminal SEP tag made of either five or nine arginines or lysines (7D12-C5R, 7D12-C9R, 7D12-C5K and 7D12-C9K, respectively). The 5-arginine and 5-lysine SEP tags increased the E. coli expression of VHH-7D12 by over 80%. Biophysical and biochemical analysis confirmed the native-like secondary and tertiary structural properties and the monomeric nature of all VHH-7D12 variants. Moreover, all VHH-7D12 variants retained a full binding activity to the EGFR extracellular domain. Finally, thermal stress with 45-minute incubation at 60 and 75 °C, where VHH-7D12 variants are unfolded, showed that the untagged VHH-7D12 formed aggregates in all of the four buffers, and the supernatant protein concentration was reduced by up to 35%. 7D12-C5R and 7D12-C9R did not aggregate in Na-acetate (pH 4.7) and Tris-HCl (pH 8.5) but formed aggregates in phosphate buffer (PB, pH 7.4) and phosphate buffer saline (PBS, pH 7.4). The lysine tags (either C5K or C9K) had the strongest solubilization effect, and both 7D12-C5K and 7D12-C9K remained in the supernatant. Altogether, our results indicate that, under a thermal stress condition, the lysine SEP tags solubilization effect is more potent than that of an arginine SEP tags, and the SEP tags did not affect the structural and functional properties of the protein.

Biophysical studies of amorphous protein aggregation and in vivo immunogenicity

“…Indeed, a successful vaccine requires, in addition to a high titer, establishment of long-term memory, production of neutralizing antibodies, lack of a cross-reaction, and needs to fulfill practical requirements for storage and handling (Lussow et al 1990 ; Powell 1996 ). In addition, we found that whereas some amorphous aggregates are highly immunogenic, others are not (Kibria et al 2021 ), strongly suggesting the need for further characterizing and classifying amorphous aggregates in classes according to biophysical properties other than size, as proposed by Narhi ( 2012 ). Nevertheless, we believe that SCP tags may be a useful tool to support the development of vaccines that use small globular proteins as immunogens without the need for additives to increase their immunogenicity (adjuvant-free).…”

“…This technique is a convenient way to increase the solubility of a protein without changing solvent conditions because the few charged amino acids added to protein termini usually do not disturb the structure and activity of the protein. As this technique was extended to other amino acid types (Islam et al 2012 ) and various proteins (Rathnayaka et al 2011 ; Nautiyal and Kuroda 2018 ; Brindha et al 2021 ; Kibria et al 2021 ; Brindha and Kuroda 2022 ), the attached peptides were named solubility-enhancing peptide tags (SEPs).

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BeStSel: webserver for secondary structure and fold prediction for protein CD spectroscopy

“…They used secondary structure analysis to compare the recombinant and the wild-type spike protein. A number of further studies involve the application of BeStSel for demonstrating that recombinant proteins have the correct secondary structure ( 24–27 ). Another purpose BeStSel is often used for is investigating the structure of antibodies ( 28–30 ).…”