title: Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling.
abstract: BACKGROUND
  Directed evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F). 3F proteins are small (~7 kDa), structurally well defined, thermally stable and resistant to proteolysis that presents them as promising candidates for directed evolution.
  
  
  RESULTS
  We have engineered a snake α-neurotoxin that belongs to the 3F family by randomizing the residues in the loops involved in binding with acetylcholine receptors and employing cDNA display to obtain modulators of interleukin-6 receptor (IL-6R). Selected candidates were highly specific for IL-6R with dissociation constants and IC50s in the nanomolar range. Antagonists as well as agonists were identified in an IL-6 dependent cell proliferation assay. Size minimization yielded peptides of about one-third the molecular mass of the original proteins, without significant loss of activities and, additionally, lead to the identification of the loops responsible for function.
  
  
  CONCLUSIONS
  This study shows 3F protein is amenable to introduce amino acid changes in the loops that enable preparation of a high diversity library that can be utilized to obtain ligands against macromolecules. We believe this is the first report of protein engineering to convert a neurotoxin to receptor ligands other than the parent receptor, the identification of an agonist from non-immunoglobulin proteins, the construction of peptide mimic of IL-6, and the successful size reduction of a single-chain protein.
authors.affiliation: National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan. mnaimuddin@gmail.com
authors.forename: Mohammed
authors.initials: M
authors.lastname: Naimuddin
authors.forename: Suzuko
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Chihiro
authors.initials: C
authors.lastname: Tsutsui
authors.forename: Masayuki
authors.initials: M
authors.lastname: Machida
authors.forename: Naoto
authors.initials: N
authors.lastname: Nemoto
authors.forename: Takafumi
authors.initials: T
authors.lastname: Sakai
authors.forename: Tai
authors.initials: T
authors.lastname: Kubo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019020:Directed Molecular Evolution; D020869:Gene Expression Profiling / Q000379:methods; D015723:Gene Library; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D019947:Receptors, Interleukin-6 / Q000819:agonists*; D016415:Sequence Alignment; D015398:Signal Transduction / Q000502:physiology*; D012910:Snake Venoms / Q000737:chemistry
chemical_list: D009498:Neurotoxins; D010455:Peptides; D019947:Receptors, Interleukin-6; D012910:Snake Venoms