Engineering Specificity from Broad to Narrow: Design of a β-Lactamase Inhibitory Protein (BLIP) Variant That Exclusively Binds and Detects KPC β-Lactamase

ACS Infect Dis. 2016 Dec 9;2(12):969-979. doi: 10.1021/acsinfecdis.6b00160. Epub 2016 Oct 26.


The β-lactamase inhibitory protein (BLIP) binds and inhibits a wide range of class A β-lactamases including the TEM-1 β-lactamase (Ki = 0.5 nM), which is widely present in Gram-negative bacteria, and the KPC-2 β-lactamase (Ki = 1.2 nM), which hydrolyzes virtually all clinically useful β-lactam antibiotics. The extent to which the specificity of a protein that binds a broad range of targets can be modified to display narrow specificity was explored in this study by engineering BLIP to bind selectively to KPC-2 β-lactamase. A genetic screen for BLIP function in Escherichia coli was used to narrow the binding specificity of BLIP by identifying amino acid substitutions that retain affinity for KPC-2 while losing affinity for TEM-1 β-lactamase. The combination of single substitutions yielded the K74T:W112D BLIP variant, which was shown by inhibition assays to retain high affinity for KPC-2 with a Ki of 0.4 nM, while drastically losing affinity for TEM-1 with a Ki > 10 μM. The K74T:W112D mutant therefore binds KPC-2 β-lactamase 3 times more tightly while binding TEM-1 > 20000-fold more weakly than wild-type BLIP. The K74T:W112D BLIP variant also exhibited low affinity (Ki > 10 μM) for other class A β-lactamases. The high affinity and narrow specificity of BLIP K74T:W112D for KPC-2 β-lactamase suggest it could be a useful sensor for the presence of this enzyme in multidrug-resistant bacteria. This was demonstrated with an assay employing BLIP K74T:W112D conjugated to a bead to specifically pull-down and detect KPC-2 β-lactamase in lysates from clinical bacterial isolates containing multiple β-lactamases.

Keywords: antibiotic resistance; diagnostics; enzyme inhibition; protein engineering; protein−protein interactions; β-lactamase.

MeSH terms

  • Bacterial Proteins / antagonists & inhibitors*
  • Bacterial Proteins / metabolism
  • Bacterial Proteins / pharmacology
  • Escherichia coli / drug effects*
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Kinetics
  • Models, Molecular
  • Protein Binding
  • Protein Engineering
  • beta-Lactamase Inhibitors / chemistry*
  • beta-Lactamase Inhibitors / metabolism
  • beta-Lactamase Inhibitors / pharmacology
  • beta-Lactamases / chemistry*
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism


  • Bacterial Proteins
  • beta-Lactamase Inhibitors
  • beta-lactamase KPC-2
  • beta-Lactamases