A suitable and effective stepwise oxidative refolding procedure for highly‐cationic tetrameric avidin in nucleic acid free conditions

Abstract

Optimized conditions are needed to refold recombinant proteins from bacterial inclusion bodies into their biologically active conformations. In this study, we found two crucial requirements for efficient refolding of cationic tetrameric chicken avidin. The first step is to eliminate nucleic acid contaminants from the bacterial inclusion body. The electrostatic interactions between the remaining nucleic acids and proteins strongly enhanced protein aggregation during the refolding process. The cysteine specific reversible S-cationization procedure was successfully employed for largescale preparation of nucleic acid free denatured protein without purification tag system. The second step is the intramolecular disulfide formation prior to refolding in dialysis removing denaturant. Disulfide intact monomeric avidin showed efficient formation of biologically active tetrameric conformation during the refolding process. Using this optimized refolding procedure, highly cationic avidin derivative designed as an intracellular delivery carrier of biotinylated protein was successfully prepared.

Hydrophobicity and molecular mass‐based separation method for autoantibody discovery from mammalian total cellular proteins

“…The TRIzol extraction procedure quantitatively extracts TCP with complete removal of nucleic acid contamination. After dissolving the extracted proteins in 6 M guanidium hydrochloride (GdnHCl), all cysteine residues were modified via the alkyl disulfide reaction, which enhanced protein solubility by introducing positive charges by TAPS‐sulfonate (Futami et al, 2015; Futami et al, 2017; Kimura et al, 2020; Miyamoto et al, 2022; Murata et al, 2006). Highly water‐soluble TCP was recovered by dialyzing against pure water without any visible precipitates.…”

“…The recovered denatured protein precipitates were solubilized in 6 M GdnHCl containing 0.1 M Tris–HCl (pH 8.5) and reduced with 30 mM dithiothreitol for 2 h at 37°C. S ‐cationization of all Cys residues in total cellular proteins was prepared by adding 70 mM [3‐(trimethylammonium)propyl] methanethiosulphonate (TAPS‐sulfonate, Katayama Chemical, Osaka, Japan) and incubating for 1 h at 37°C (Futami et al, 2015; Futami et al, 2017; Kimura et al, 2020; Miyamoto et al, 2022; Murata et al, 2006). Total cellular protein, with or without S ‐cationization, was adjusted to a protein concentration of 3 mg/mL with 6 M GdnHCl and mixed with a 0.1 volume of acetic acid.…”

“…To digest nucleic acids, 125 unit/ mL of Benzonase (Merck Millipore, Burlington, MA, USA) were added and incubated for 30 min at 25 C. Nucleic acid-free, fully denatured, and water-soluble total cellular proteins were prepared using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) according to previously described methods (Futami et al, 2014). The recovered denatured protein precipitates were solubilized in 6 M GdnHCl containing 0.1 M Tris-HCl (pH 8.5) and reduced with 30 mM dithiothreitol for 2 h at 37 C. Scationization of all Cys residues in total cellular proteins was prepared by adding 70 mM [3-(trimethylammonium) propyl] methanethiosulphonate (TAPS-sulfonate, Katayama Chemical, Osaka, Japan) and incubating for 1 h at 37 C (Futami et al, 2015;Futami et al, 2017;Kimura et al, 2020;Miyamoto et al, 2022;Murata et al, 2006). Total cellular protein, with or without Scationization, was adjusted to a protein concentration of 3 mg/mL with 6 M GdnHCl and mixed with a 0.1 volume of acetic acid.…”

Engineering Cancer/Testis Antigens With Reversible S-Cationization to Evaluate Antigen Spreading

“…In order to quantitatively evaluate antigen spreading, we designed a multiple S- cationized antigen-immobilized bead array (MUSCAT) assay system ( 33 ). S- cationization techniques are employed as a powerful solubilization tool by conjugation of cationic moieties in sulfhydryl groups in denatured protein ( 34 – 36 ). Full-length and water-soluble S- cationized antigens were covalently immobilized onto Luminex magnetic beads via the activated carboxylic acid of the COOH radical group ( 33 , 37 ).…”