Convenient and efficient in vitro folding of disulfide-containing globularprotein from crude bacterial inclusion bodies

We investigated how the folding yield of disulfide-containing globular prot eins having positive net charges from crude bacterial inclusion bodies was affected by additives in the folding buffer. In screening folding condition s for human ribonucleases and its derivative, we found that addition of sal t (about 0.4 M) to a folding buffer increased the folding yield. This sugge sted that electrostatic interaction between polyanionic impurities such as nucleic acids and cationic unfolded protein led to the formation of aggrega tes under the low-salt conditions. Since inclusion bodies were found to con tain nucleic acids regardless of the electrostatic nature of the expressed protein, the electrostatic interaction between phosphate moieties of nuclei c acids and basic amino acid residues of a denatured protein may be large e nough to cause aggregation, and therefore the addition of salt in a folding buffer may generally be useful for promotion of protein folding from crude inclusion bodies. We further systematically investigated additives such as glycerol, guanidium chloride, and urea that are known to act as chemical c haperons, and found that these additives, together with salt, synergistical ly improved folding yield. This study, suggesting that the addition of salt into the folding buffer is one of the crucial points to be considered, may pave the way for a systematic investigation of the folding conditions of d isulfide-containing foreign proteins from crude bacterial inclusion bodies.