Variants of human growth hormone (hGH) with increased affinity and specificity for the hGH receptor were isolated using an improved phage display system. Nearly one million random mutants of hGH were generated at 12 sites previously shown to modulate binding to the hGH receptor or human prolactin (hPRL) receptor. The mutant hormones were displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. After three to six cycles of enrichment for hGH-phage particles that bound to hGH receptor beads, we isolated hGH mutants that exhibited consensus binding sequences for the hGH receptor. Residues previously identified as important for hGH receptor binding by alanine-scanning mutagenesis were more highly conserved by this selection method. However, other residues nearby were not optimal, and by mutating them, hormone variants having greater affinity and selectivity for the hGH receptor were isolated. This approach should be useful for those who wish to modify and understand the energetics of protein-ligand interfaces.

In both panels, the columns labeled "⌬espIϩpespI" should read "⌬espI" and the columns labeled "⌬espI" should read "⌬espIϩpespI."

The infiltration and activation of monocytes is a hallmark of chronic inflammation, including that associated with a variety of disease states such as rheumatoid arthritis, atherosclerosis, and various autoimmune conditions. Recently, a family of small molecular mass proteins has been described which appear to have inflammatory properties, including chemoattractant effects on monocytes. We report here on the molecular cloning, characterization, and functional expression of mu RANTES, a new murine member of this family. mu RANTES expressed in a mammalian expression system is an approximately 8-kDa protein exhibiting immune cross-reactivity with a rabbit polyclonal antiserum generated against human RANTES. Boyden chamber chemotaxis experiments reveal some lack of species specificity in monocyte chemoattractant potential, as recombinant mu RANTES attracts human monocytes in a dose-dependent fashion in vitro. mu RANTES and its human homolog share approximately 85% amino acid identity, a higher level of conservation than that seen with any other species homologs in this cytokine family, and second only to transforming growth factor-beta among reported immune cytokines.

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