RECOMBINANT APROTININ HOMOLOG WITH NEW INHIBITORY SPECIFICITY FOR CATHEPSIN-G

The substitution of amino acids in the reactive site of aprotinin, a bovine serine proteinase inhibitor with potent activity against trypsin, plasmin and tissue kallikrein, led to a change in specificity of the inhibitor. Twelve new aprotinin variants prepared by recombinant DNA technology and expressed in Escherichia coli clearly demonstrated that the neighbouring groups of the P1 residue, in particular P'2, contribute to the specificity of the inhibitor, while earlier investigations on semisynthetically prepared variants revealed the importance of the P1 residue in dominating the inhibitory specificity. Recombinant aprotinin variants which act specifically against chymotrypsin-like proteinases, were obtained by substitution of the amino acids in position P1 and P'2 by hydrophobic amino acids like phenylalanine, tyrosine and leucine. Some of these variants, particularly those with phenylalanine or leucine substitutions, were also found to exhibit inhibitory activity against cathepsin G with an equilibrium constant of dissociation K(i) of 10(-8) M. Inhibitory specificity against cathepsin G was not found in any semisynthetic variant prepared earlier.