Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in <Emphasis Type="Italic">Escherichia coli</Emphasis> |
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Authors: | Shaofang Liu Yingjie Chen Yandu Lu Huaxin Chen Fuchao Li Song Qin |
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Institution: | (1) Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 266071 Qingdao, People’s Republic of China;(2) Graduate School, Chinese Academy of Sciences, 100049 Beijing, People’s Republic of China;(3) Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, 264003 Yantai, People’s Republic of China |
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Abstract: | 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 α and β 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. |
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