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-amino acid substitutions were constructed at each of 105 positions in the gene for hIL-3 . Approximately eight single-site substitutions at each position were produced in osmotic shock fractions and screened for activity. 15 mutants were found with bioactivity of 5-26-fold greater than that of native hIL-3. The majority of amino acids in hIL-3 15-125 could be substituted without substantial loss of activity. Substitution of residues predicted to be in the hydrophobic core of the protein often resulted in reduced activity and/or low accumulation levels. 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 the receptor.Human interleukin-3 (hIL-3) 1 is a multilineage hematopoietic cytokine acting in the bone marrow to promote the growth of most lineages of blood cell precursors (1). Recently, exogenously administered hIL-3 has shown promise for the clinical relief of neutropenia and thrombocytopenia induced by cancer chemotherapy (2, 3). Sequence homology comparisons of hIL-3 with other proteins indicate that it is a member of the hematopoietic cytokine family (4 -6) and that it adopts a four-␣-helix bundle topology (7-10). The protein binds to a receptor comprising at least two nonidentical subunits (11, 12). Although the precise nature of interaction between hIL-3 and its receptor is not known, studies using site-specific mutants have shed some light on which portions of the protein are important for function (8,(13)(14)(15)(16)(17). In particular, mutagenesis of the adjacent helices A and D indicate that these regions are important for interaction with the receptor. This is similar to the findings for human interleukin-5 and human granulocyte-macrophage colony stimulating factor, whose receptors share a common ␤ subunit with the hIL-3 receptor (11,18,19). Other members of the hematopoietic cytokine family also have important residues in helices A and D (19 -23) and in helix C (20,22,24,25).In this paper we have undertaken an extensive mutagenesis of hIL-3 in order to discover mutants with enhanced proliferative activity and to define residues necessary for activity. Although alanine scanning mutagenesis has been successfully used to derive structure-activity information (20, 26 -32), we chose to perform a more extensive mutagenesis, permitting the incorporation of any of the possible 19 substitutions (33). MATERIALS AND METHODS Production of hIL-3 and Variants in the Escherichia coli Cytoplasm-General techniques for manipulation of DNA are described elsewhere (34). The hIL-3 gene (35) was obtained from British Biotechnolo...