Bacterial surface display library screening by target enzyme labeling: Identification of new human cathepsin G inhibitors

Anal Biochem. 2005 Nov 15;346(2):258-67. doi: 10.1016/j.ab.2005.08.019. Epub 2005 Sep 1.

Abstract

The aim of this study was to establish a new tool for screening surface displayed peptide libraries based on the idea that cells expressing an enzyme inhibitor at the surface can be specifically labeled by the target enzyme. For this purpose peptide P15, exhibiting a K(i) value of 0.25 microM toward human cathepsin G, was expressed on the Escherichia coli cell surface by the use of Autodisplay. Purified cathepsin G was coupled to biotin and incubated with cells expressing the inhibitor. After addition of streptavidin-fluorescein isothiocyanate, these cells could be clearly differentiated from control cells by whole-cell fluorescence using flow cytometer analysis. To determine whether this protocol can be used for the sorting of single cells, a mixed population of cells with and without inhibitor was treated accordingly. Single cells were selected by increased fluorescence and sorted using fluorescence-activated cell sorting (FACS). Single cell clones were obtained and subjected to DNA sequence analysis. It turned out that the bacteria selected by this protocol displayed the correct peptide inhibitor at the cell surface. The protocol was then used to screen random peptide libraries, expressed at the cell surface, and a new lead structure for human cathepsin G (IC50 = 11.7 microM) was identified. The new drug discovery tool presented here consists of three steps: (a) surface display of peptide libraries, (b) selection of single cells with inhibiting structures by using the inherent affinity of the target enzyme, and (c) sorting of single cells, which were labeled by FACS.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Outer Membrane Proteins / genetics
  • Cathepsin G
  • Cathepsins / antagonists & inhibitors*
  • Cathepsins / metabolism
  • Epitopes / genetics
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Flow Cytometry / methods
  • Humans
  • Microscopy, Fluorescence
  • Microscopy, Phase-Contrast
  • Molecular Sequence Data
  • Peptide Library*
  • Peptides / genetics*
  • Recombinant Fusion Proteins / genetics
  • Serine Endopeptidases / metabolism
  • Serine Proteinase Inhibitors / isolation & purification*
  • Serine Proteinase Inhibitors / metabolism

Substances

  • Bacterial Outer Membrane Proteins
  • Epitopes
  • Peptide Library
  • Peptides
  • Recombinant Fusion Proteins
  • Serine Proteinase Inhibitors
  • glutamyl-isoleucyl-leucyl-isoleucyl-phenylalanyl-tryptophyl-seryl-lysyl-aspartyl-isoleucyl-glycyl-tyrosyl-seryl-phenylalanyl-threonine
  • Cathepsins
  • Serine Endopeptidases
  • CTSG protein, human
  • Cathepsin G