Preparation and application to protein engineering of thiol-protecting reagent having charges.

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In order to enhance the utility in protein engineering of a system using Escherichia coli producing extraneous disulfide-containing proteins as inclusion bodies, we attempted to prepare thiol-protecting reagents having charges that can solubilize denatured proteins, by reversible modification of SH groups in reduced proteins, without using denaturants. For preliminary experiments, we reduced disulfides of four proteins and alkylated the SH groups with charged S-alkylating reagents, and examined the relationship between the solubilities and the values of net charge per hydrophobic residue of the resultant denatured proteins. As a result, we found that a denatured protein is well soluble in water (more than 1mg/ml) when the above value is more than +0.17 or less than -0.32. Since S-alkylation is not reversible, we next prepared alkyl methanethiosulfonate derivatives which possess charges in the alkyl moieties and can react with SH groups to intorduce charges into reduced proteins as mixe … More d disulfides (S-alkylsulfenylation). Reduced protein modified with these reagents indicated that the denatured protein became soluble when basic protein was modified with a positively charged reagent, trimethylammoniopropyl methanethiosulfonate (TAPS-sulfonate), and acidic one with negatively charged reagent, as expected. Mixed disulfide groups thus introduced in the reduced protein were enough stable under the conditions of proteolysis, which is required for N-terminal processing in protein engineering, but could be easily reverted back to SH groups by reduction. Furthermore, TAPS-sulfonate could be successfully applied to solubilize and extract recombinant human RNase 4 and the secreted form of human fibroblast growth factor receptor, both of which were produced in E.coli as inclusion bodies and had not been able to be extracted form cell debris by means of the previous methods because of their insolubility. Purified denatured RNase 4 (TAPS-RNase 4) could be folded into the active structure by SH-SS interchange reaction with a glutathione redox system. All of these results indicate that TAPS-sulfonate prepared here is very useful for extraction, N-terminal processing and folding of disulfide-containing proteins extraneously produced in E.coli as inclusion bodies. Less