In vitro refolding of porcine pepsin immobilized on agarose beads

Since in vitro refolding of pepsin has long been attempted without success, it has been suspected that pepsin has no intrinsic refolding ability. In t he present study, in order to eliminate unfavorable intermolecular interact ions bringing about aggregation and autoproteolysis, we immobilized pepsin onto agarose beads. This technique enabled us to search extensively for app ropriate refolding conditions without limitation of the refolding period. R enaturation of immobilized pepsin was observed exclusively at pH 3-5. This process was extremely slow and reached equilibrium after 300 h. Sixty perce nt of the proteolytic activity was recovered at pH 5. Addition of salts rai sed the recovery to 80% but had no significant effect on the refolding rate , suggesting that the salts mainly stabilize the native state of pepsin. Th is is the first report on the successful in vitro refolding of pepsin.