Abstract
Human immunoglobulin heavy chain variable domains (VH) are promising scaffolds for antigen binding. However, VH is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the VH domain, we performed in vivo protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of E. coli. After screening a human germ-line VH library, 95% of the VH proteins obtained were identified as VH3 family members; one VH protein, MG2x1, stood out among separate clones expressing individual VH variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in VH. Comparison of the crystal structures of the VH variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the VH interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered VH acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the VH variant as a scaffold was qualified as most of VH clones selected randomly were expressed as soluble form in E. coli regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected VH conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display.
Highlights
The variable domain of heavy or light chain (VH or VL) of a human immunoglobulin G (IgG) molecule is the smallest part of the antibody that preserves the original binding activity
Verifying Tat-associated protein engineering (TAPE) To verify whether TAPE system can discriminate between proteins of different solubilities, we applied this system to various published VH domains whose soluble expression levels are well known
The VH domains were cloned into the pET-TAPE vector (Figure 1A), allowing them to be expressed in E. coli as fusions with BLA and the Tat signal sequence of signal sequence of trimethylamine N-oxide reductase (ssTorA)
Summary
The variable domain of heavy or light chain (VH or VL) of a human immunoglobulin G (IgG) molecule is the smallest part of the antibody that preserves the original binding activity. Variable domains have short serum half-lives and lack effector function, their format flexibility by adopting immune cell engaging strategy or introducing a long-acting module can ameliorate these defects [1,2,3] Their ability to access occluded or hidden epitopes, superior bio-distribution, and cost-effective production make variable domains potentially useful in therapeutic applications for which full IgG molecules are not appropriate [4,5,6]. When not assembled with each other, instability problem of VH and VL of human IgG is a major concern for biotechnological applications since Ward et al reported that such VH domains are relatively sticky resulting in tendency to aggregate [7] This aggregation is primarily due to interactions between hydrophobic patches residing at the interface between VH and VL. Three hydrophilic substitutions (G44E/L45R/W47G) improve the solubility of VH [8,9,10], but these changes decrease expression yield and thermal stability due to the resultant deformations of the b-sheet structure [11,12,13]
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