title: Engineering human prolactin to bind to the human growth hormone receptor.
abstract: A strategy of iterative site-directed mutagenesis and binding analysis was used to incorporate the receptor-binding determinants from human growth hormone (hGH) into the nonbinding homolog, human prolactin (hPRL). The complementary DNA for hPRL was cloned, expressed in Escherichia coli, and mutated to introduce sequentially those substitutions from hGH that were predicted by alanine-scanning mutagenesis and other studies to be most critical for binding to the hGH receptor from human liver. After seven rounds of site-specific mutagenesis, a variant of hPRL was obtained containing eight mutations with an association constant for the hGH receptor that was increased more than 10,000-fold. This hPRL variant binds one-sixth as strongly as wild-type hGH, but shares only 26 percent overall sequence identity with hGH. These studies show the feasibility of recruiting receptor-binding properties from distantly related and functionally divergent hormones and show that a detailed functional database can be used to guide the design of a protein-protein interface in the absence of direct structural information.
authors.affiliation: Department of Protein Engineering, Genentech, Inc. South San Francisco, CA 94080.
authors.forename: B C
authors.initials: BC
authors.lastname: Cunningham
authors.forename: D J
authors.initials: DJ
authors.lastname: Henner
authors.forename: J A
authors.initials: JA
authors.lastname: Wells
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D013006:Growth Hormone / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011388:Prolactin / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011986:Receptors, Somatotropin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D011388:Prolactin; D013006:Growth Hormone