PMID- 10051566
OWN - NLM
STAT- MEDLINE
DCOM- 19990415
LR  - 20190501
IS  - 0027-8424 (Print)
IS  - 1091-6490 (Electronic)
IS  - 0027-8424 (Linking)
VI  - 96
IP  - 5
DP  - 1999 Mar 2
TI  - Small antibody-like proteins with prescribed ligand specificities derived from 
      the lipocalin fold.
PG  - 1898-903
AB  - We demonstrate that the ligand pocket of a lipocalin from Pieris brassicae, the 
      bilin-binding protein (BBP), can be reshaped by combinatorial protein design such 
      that it recognizes fluorescein, an established immunological hapten. For this 
      purpose 16 residues at the center of the binding site, which is formed by four 
      loops on top of an eight-stranded beta-barrel, were subjected to random 
      mutagenesis. Fluorescein-binding BBP variants were then selected from the mutant 
      library by bacterial phage display. Three variants were identified that complex 
      fluorescein with high affinity, exhibiting dissociation constants as low as 35.2 
      nM. Notably, one of these variants effects almost complete quenching of the 
      ligand fluorescence, similarly as an anti-fluorescein antibody. Detailed 
      ligand-binding studies and site-directed mutagenesis experiments indicated (i) 
      that the molecular recognition of fluorescein is specific and (ii) that charged 
      residues at the center of the pocket are responsible for tight complex formation. 
      Sequence comparison of the BBP variants directed against fluorescein with the 
      wild-type protein and with further variants that were selected against several 
      other ligands revealed that all of the randomized amino acid positions are 
      variable. Hence, a lipocalin can be used for generating molecular pockets with a 
      diversity of shapes. We term this class of engineered proteins "anticalins." 
      Their one-domain scaffold makes them a promising alternative to antibodies to 
      create a stable receptor protein for a ligand of choice.
FAU - Beste, G
AU  - Beste G
AD  - Abteilung Proteinchemie, Institut fur Biochemie, Technische Universitat 
      Darmstadt, Germany.
FAU - Schmidt, F S
AU  - Schmidt FS
FAU - Stibora, T
AU  - Stibora T
FAU - Skerra, A
AU  - Skerra A
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Proc Natl Acad Sci U S A
JT  - Proceedings of the National Academy of Sciences of the United States of America
JID - 7505876
RN  - 0 (Antibodies, Catalytic)
RN  - 0 (Carrier Proteins)
RN  - 0 (Insect Hormones)
RN  - 0 (Insect Proteins)
RN  - 0 (Ligands)
RN  - 0 (Oligodeoxyribonucleotides)
RN  - 0 (Recombinant Proteins)
RN  - 0 (bilin-binding protein, insect)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Antibodies, Catalytic/*chemistry/metabolism
MH  - Base Sequence
MH  - Binding Sites
MH  - Carrier Proteins/*chemistry/metabolism
MH  - Cloning, Molecular
MH  - Gene Library
MH  - Insect Hormones
MH  - *Insect Proteins
MH  - Insecta
MH  - Ligands
MH  - Models, Molecular
MH  - Molecular Sequence Data
MH  - Mutagenesis, Insertional
MH  - Mutagenesis, Site-Directed
MH  - Nuclear Magnetic Resonance, Biomolecular
MH  - Oligodeoxyribonucleotides/chemical synthesis/chemistry
MH  - Protein Folding
MH  - Protein Structure, Tertiary
MH  - Recombinant Proteins/chemistry/metabolism
PMC - PMC26708
EDAT- 1999/03/03 00:00
MHDA- 1999/03/03 00:01
PMCR- 1999/09/02
CRDT- 1999/03/03 00:00
PHST- 1999/03/03 00:00 [pubmed]
PHST- 1999/03/03 00:01 [medline]
PHST- 1999/03/03 00:00 [entrez]
PHST- 1999/09/02 00:00 [pmc-release]
AID - 4821 [pii]
AID - 10.1073/pnas.96.5.1898 [doi]
PST - ppublish
SO  - Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):1898-903. doi: 10.1073/pnas.96.5.1898.