DESIGN AND CHARACTERIZATION OF LONG-R(3)-INSULIN-LIKE GROWTH-FACTOR-IMUTEINS WHICH SHOW RESISTANCE TO PEPSIN DIGESTION

Site-directed mutagenesis was used to construct pepsin-resistant, sing le-point mutations of the N-terminal extended IGF-I analogue, long-R(3 )-IGF-I. In order to identify the most susceptible sites, the kinetics of long-R(3)-IGF-I digestion by purified porcine pepsin were determin ed. Pepsin initially cleaved the Leu10-Phe11 bond in the N-terminal ex tension peptide to generate FVN-R(3)-IGF-I, followed in rapid successi on by cleavage at Gln15-Phe16, Tyr24-Phe25, Leu10-Val11 and Met59-Tyr6 0 in the IGF-I moiety. Single-point mutations at these sites were desi gned on the basis of the preferred cleavage bonds for pepsin, as well as amino acid substitutions less likely to disturb protein structure. These included Leu10Val, Phe16Ala, Phe25Leu, Asp53Glu and Met59Gln. Al l five muteins retained growth-promoting activity equivalent to or hig her than that of IGF-I. In terms of pepsin susceptibility, Leu10Val an d Asp53Glu were degraded as rapidly as the parent long-R(3)-IGF-I, Met 59Gln and Phe25Leu were partially stabilised, and Phe16Ala showed a ma rked improvement in stability over a wide range of pepsin:substrate ra tios. Accordingly, the Phe16Ala mutein, long-R(3)A(16)-IGF-I, has pote ntial for oral applications to enhance gastric growth and repair.