Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli.
Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC alpha and beta subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.
Liu
Shaofang
S
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 266071 Qingdao, People's Republic of China. lsf909@sina.com
Chen
Yingjie
Y
Lu
Yandu
Y
Chen
Huaxin
H
Li
Fuchao
F
Qin
Song
S
D000042:Absorption; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D000458:Cyanobacteria / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010798:Phycocyanin / Q000096:biosynthesis* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D011994:Recombinant Proteins; C002497:allophycocyanin; D010798:Phycocyanin