title: Glubodies: randomized libraries of glutathione transferase enzymes.
abstract: BACKGROUND
  The immunoglobulin framework has been mutagenized to engineer recombinant libraries of proteins as potential diagnostics and novel catalysts, although the often shallow binding cleft may limit the utility of this framework for binding diverse small organic molecules. By contrast, the glutathione S-transferase (GST) family of enzymes contains a deep binding cleft, which has evolved to accommodate a broad range of hydrophobic xenobiotics. We set out to determine whether GST molecules with novel ligand-binding characteristics could be produced by random mutagenesis of segments of the binding cleft.
  
  
  RESULTS
  We have identified two ligand-recognition segments (LRSs) in human GST P1, which are near the active site in the folded protein, but have characteristics indicating that the integrity of their sequence is not essential for the overall structure or activity of the protein. Libraries of GST P1-derived proteins were produced by substituting randomized sequences for an LRS or inserting random sequences into an LRS. The recombinant proteins in the libraries, collectively designated as 'glubodies,' generally retain enzymatic activity but differ markedly both from each other and from the parent enzyme in sensitivity to inhibition by diverse small organic compounds. In some instances, a glubody is inhibited by completely novel structures.
  
  
  CONCLUSIONS
  We have shown that a non-antibody framework can be used to create large libraries of proteins with a wide range of binding specificities for small organic molecules. The glubodies provide a rich source of data for correlating the structural and functional features of proteins relevant to ligand binding. The criteria applied for identifying an LRS in GST P1 are generally applicable to other protein frameworks.
authors.affiliation: Terrapin Technologies, Incorporated, 750 Gateway Boulevard, South San Francisco, CA 94080, USA. gnap@trpntech.com
authors.forename: E W
authors.initials: EW
authors.lastname: Napolitano
authors.forename: H O
authors.initials: HO
authors.lastname: Villar
authors.forename: L M
authors.initials: LM
authors.lastname: Kauvar
authors.forename: D L
authors.initials: DL
authors.lastname: Higgins
authors.forename: D
authors.initials: D
authors.lastname: Roberts
authors.forename: J
authors.initials: J
authors.lastname: Mandac
authors.forename: S K
authors.initials: SK
authors.lastname: Lee
authors.forename: A
authors.initials: A
authors.lastname: Engqvist-Goldstein
authors.forename: R
authors.initials: R
authors.lastname: Bukar
authors.forename: B L
authors.initials: BL
authors.lastname: Calio
authors.forename: H M
authors.initials: HM
authors.lastname: Jäck
authors.forename: J A
authors.initials: JA
authors.lastname: Tainer
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D005982:Glutathione Transferase