Overproduction of Thermus sp YS 8-13 manganese catalase in Escherichia coli - Production of soluble apoenzyme and in vitro formation of active holoenzyme

Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia col i BL21(DE3) was accomplished by introducing a derivative of pET-23a(+) cont aining a copy of the coding gene into the multicloning site. E. coli BL21(D E3)/pETMNCAT produced abundant quantities of manganese catalase as insolubl e inclusion bodies. Regeneration of active catalase was achieved by denatur ation in guanidine hydrochloride and subsequent dialysis in the presence of manganese ion. When the E. coli chaperone genes GroEL, GroES, DnaK, DnaJ a nd GrpE were coexpressed with manganese catalase, a significant fraction of the overproduced protein was partitioned into the soluble fraction. Howeve r, almost all of the soluble enzyme was isolated in a manganese-deficient a po form which could subsequently be converted into active holoenzyme by inc ubation with manganese ion at high temperatures. Further experiments on thi s apo catalase suggested that the structure of this protein was virtually i dentical to the active holoenzyme.