Mutations for decreasing the immunogenicity and maintaining the function of core streptavidin

Abstract

The defining property of core streptavidin (cSA) is not only its high binding affinity for biotin but also its pronounced thermal and chemical stability. Although potential applications of these properties including therapeutic methods have prompted much biological research, the high immunogenicity of this bacterial protein is a key obstacle to its clinical use. To this end, we have successfully constructed hypoimmunogenic cSA muteins in a previous report. However, the effects of these mutations on the physicochemical properties of muteins were still unclear. These mutations retained the similar electrostatic charges to those of wild-type (WT) cSA, and functional moieties with similar hydrogen bond pattern. Herein, we performed isothermal titration calorimetry, differential scanning calorimetry, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to gain insight into the physicochemical properties and functions of these modified versions of cSA. The results indicated that the hypoimmunogenic muteins retained the biotin-binding function and the tetramer structure of WT cSA. In addition, we discuss the potential mechanisms underlying the success of these mutations in achieving both immune evasion and retention of function; these mechanisms might be incorporated into a new strategy for constructing hypoimmunogenic proteins.

Disruption of cell adhesion by an antibody targeting the cell-adhesive intermediate (X-dimer) of human P-cadherin

“…Antibodies were eluted with 10 mL of elution buffer (20 mM Tris, 500 mM NaCl, 300 mM imidazole, 6 M Guanidine-HCl, pH 8.0). The antibodies were then refolded by the step-wise dilution method described previously36. Finally, the refolded antibodies were purified by size exclusion chromatography (SEC) on a Hiload 26/60 Superdex 75 column equilibrated with buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM CaCl 2 , pH 7.5.…”

Cancer nanotechnology: The impact of passive and active targeting in the era of modern cancer biology

“…It is particularly useful to establish proof of concepts or to screen different ligands without interference from the conjugation chemistry or to study fundamental ligand decoration parameters [185, 186]. The weakness of this method is that it may cause immunogenicity due to the presence of the exogenous protein on the surface and it is not usually suitable for human use [187]. Also, because avidin can bind biotin in a multivalent fashion, cross-linking between the NP constituents can also occur.…”

Structure-based design and synthesis of a bivalent iminobiotin analog showing strong affinity toward a low immunogenic streptavidin mutant

“…212 (V212), that overcame the two problems outlined above. Briefly, we first developed a low immunogenic SA mutant, LISA-314 by carrying out six amino acid substitutions (Y22S/ Y83S/R84 K/E101D/R103 K/E116 N) to residues in wild-type SA (SA-WT) in its immune recognition site, 21) and determined its crystal structure (Fig. S1A).…”