Cell-free production of Gaussia princeps luciferase--antibody fragment bioconjugates for ex vivo detection of tumor cells

Biochem Biophys Res Commun. 2009 Dec 18;390(3):971-6. doi: 10.1016/j.bbrc.2009.10.087. Epub 2009 Oct 21.


Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Neoplasm / biosynthesis*
  • Antibodies, Neoplasm / genetics
  • Antibodies, Neoplasm / immunology
  • Copepoda / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Humans
  • Immunoglobulin Idiotypes / biosynthesis
  • Immunoglobulin Idiotypes / genetics
  • Immunoglobulin Idiotypes / immunology
  • Immunoglobulin Variable Region / biosynthesis*
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / immunology
  • Luciferases / biosynthesis*
  • Luciferases / genetics
  • Luciferases / immunology
  • Lymphoma, B-Cell / diagnosis*
  • Mice
  • Molecular Sequence Data
  • Protein Biosynthesis
  • Protein Engineering*
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology


  • Antibodies, Neoplasm
  • Immunoglobulin Idiotypes
  • Immunoglobulin Variable Region
  • Recombinant Fusion Proteins
  • Luciferases