(204h) Small-Molecular Foldase Mimics Facilitating Oxidative Protein Refolding

Oxidative protein folding is not only relevant to some diseases in vivo, but also turns out to be a bottleneck in the production of many disulfide-containing recombinant proteins expressed as inclusion bodies. Protein disulfide isomerases (PDI) and DsbA are important foldases catalyzing protein disulfide formation in vivo. To facilitate in vitro oxidative protein refolding, several small molecular artificial foldases have been designed, but they only focused on mimicking the CXXC sequence motifs in the enzymes. Herein, based on the structural characteristic of PDI and DsbA that have hydrophobic regions around the active sites, hydrophobic alkyl tails are linked to cystamine to create new small molecular foldase mimics, acyl (n-octanoyl, n-hexanoyl, and n-butanoyl) cystamine. Both the oxidizing power and oxidation specificity of cystamine are enhanced by n-octanoyl or n-hexanoyl tail. N-octanoyl and n-hexanoyl cystamine are very effective to facilitate oxidative protein refolding at strong reducing environments. Thus, with them as the oxidants, the necessity to remove excess DTT in the reduced and denatured protein solutions can be greatly alleviated. Because n-hexanoyl cystamine has moderate hydrophobicity, it is promising for application in oxidative protein refolding at an extensive concentration range. It is observed that in the oxidative refolding of 0.2 mg/mL lysozyme and ribonuclease A, only about half of n-hexanoyl cystamine is needed as compared to cystamine to achieve the same kinetic effect.