In a previous study, random-sequence proteins of 120-130 amino acid residues mere inserted into the surface loop region of the enzyme, Escherichia coli RNase HI [Doi et al. (1997) FEBS Lett. 102, 177-180]. Here we established that the RNase H activity of the insertion mutants is correlated with their secondary structure contents evaluated by circular dichroism measurement at 222 nm. The random-sequence insert of a mutant enzyme possessing relatively high RNase H activity was detached from the RNase HI scaffold, and its characterization indicated that the random-sequence protein maintains its secondary structure after separation from the scaffold. Thus, the structural features of random-sequence proteins were suggested to be monitored by measuring the activity of the scaffold enzyme into which these proteins have been inserted.
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