Development of Efficient Protein Refolding Systems Using Chaperonins

摘要

Application of chaperonins to in vitro protein refolding was investigated. E. coli chaperonin GroEL/ES and Thermus thermophilus holo-chaperonin (T. thermophilus holo-cpn) were used for construction of protein refolding systems. The systems based on immobilized chaperonin, fusion chaperonin and chaperonin in combination with ultrafiltration were chaperonin, fusion chaperonin and chaperonin in combination with ultrafiltration were chaperonin, fusion chaperonin and chaperonin in combination with ultrafiltration were investigated. Fusion chaperonin with the hexa-histidine affinity tag (GroEL-(His)_6) was efficiently purified by affinity column. The refolding activity of GroEL-(His)_6) was proved to be similar to that of native one. GroEL/ES was immobilized by covalent bond. In addition, GroEL-(His)_6 was immobilized on metal chelate resin by affinity interaction. These immobilized chaperonins retained sufficiently high refolding activities and were reused efficiently. Especially, immobilized GroEL-(His)_6 showed a high refolding activity. On the other hand, in ultrafiltration system, T. thermophilus holo-cpn, which forms a stable complex, was efficiently separated from refolded proteins and repeatedly used for protein refolding. The selection of the system is mainly dependent on the type of chaperonin most effective for the refolding of target proteins.