Biosynthesis of fluorescent allophycocyanin alpha-subunits by autocatalysis in Escherichia coli.
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.
Zhang
Weijie
W
Institute of Oceanology, Key Laboratory of Experimental Marine Biology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, People's Republic of China.
Guan
Xiangyu
X
Yang
Yu
Y
Ge
Baosheng
B
Chen
Huaxin
H
Li
Fuchao
F
Qin
Song
S
D000042:Absorption; D055162:Biocatalysis; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors; D006639:Histidine; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D008670:Metals; D010084:Oxidation-Reduction; D052980:Phycobilins / Q000235:genetics / Q000378:metabolism; D010798:Phycocyanin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic
D016166:Free Radical Scavengers; D008164:Luminescent Proteins; D008670:Metals; D052980:Phycobilins; D011994:Recombinant Proteins; C002497:allophycocyanin; D010798:Phycocyanin; C011069:phycocyanobilin; D006639:Histidine