Biosynthesis of Fluorescent Allophycocyanin Alpha-Subunits by Autocatalysis in Escherichia Coli

Biotechnol Appl Biochem. 2009 Feb;52(Pt 2):135-40. doi: 10.1042/BA20070187.

Abstract

APC (allophycocyanin) is widely used for fluorescence tagging and may be a promising antioxidant agent for use within the food and pharmaceutical industries. Chromophore attachment to apo-ApcA (apo-APC alpha-subunit without chromophore) can be autocatalysed both in vitro and in vivo. In the present study, a plasmid containing genes of apo-ApcA and chromophore synthetases [HO1 (ferredoxin-dependent haem oxygenase) and PcyA (phycocyanobilin:ferredoxin oxidoreductase)] was constructed and expressed in Escherichia coli. The results show that holo-ApcA (APC alpha-subunit with chromophore) can be synthesized by autocatalysis in E. coli. Recombinant holo-ApcA showed the same spectral and fluorescent properties as PC (phycocyanin) and could serve as a good substitute for native PC for fluorescent tagging. Moreover, recombinant ApcA can inhibit hydroxyl and peroxyl radicals more strongly than holo-ApcA and native APC. The EC(50) values were 296.4+/-22.4 microg/ml against hydroxyl radicals and 38.5+/-2.6 microg/ml against peroxyl radicals.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Absorption
  • Biocatalysis*
  • Blotting, Western
  • Chromatography, Affinity
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Free Radical Scavengers / metabolism
  • Free Radical Scavengers / pharmacology
  • Gene Expression
  • Genetic Vectors
  • Histidine
  • Luminescent Proteins / biosynthesis*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Luminescent Proteins / pharmacology
  • Metals
  • Oxidation-Reduction
  • Phycobilins / genetics
  • Phycobilins / metabolism
  • Phycocyanin / biosynthesis*
  • Phycocyanin / genetics
  • Phycocyanin / metabolism
  • Phycocyanin / pharmacology
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / pharmacology
  • Spectrometry, Fluorescence
  • Transformation, Genetic

Substances

  • Free Radical Scavengers
  • Luminescent Proteins
  • Metals
  • Phycobilins
  • Recombinant Proteins
  • allophycocyanin
  • Phycocyanin
  • phycocyanobilin
  • Histidine