SATURATION MUTAGENESIS OF HUMAN INTERLEUKIN-3

A deletion variant of human interleukin-3, hIL-3(15-125), was produced in the periplasmic space of Escherichia coli and had full activity in an AML193.1.3 cell proliferation assay. Libraries of random single-am ino acid substitutions were constructed at each of 105 positions in th e gene for hIL-3(15-125). Approximately eight single-site substitution s at each position were produced in osmotic shock fractions and screen ed for activity, 15 mutants were found with bioactivity of 5-26-fold g reater than that of native hIL-3. The majority of amino acids in hIL-3 (15-125) could be substituted without substantial loss of activity. Su bstitution of residues predicted to be in the hydrophobic core of the protein often resulted in reduced activity and/or low accumulation lev els. Only five residues predicted to be on the surface of the protein were intolerant of substitution and hence are candidates for sites of interaction with the receptor. We therefore propose that the majority of residues in hIL-3 serve a structural role and permit the display of a few key residues in the correct configuration for recognition by th e receptor.