title: Biosynthesis, spectral properties and thermostability of cyanobacterial allophycocyanin holo-α subunits.
abstract: Allophycocyanin (APC) is generally used as fluorescent labels. In this study, apcA genes from a mesophilic cyanobacterium Synechocystis sp. PCC 6803 and a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 were cloned into expression vectors to construct pathway for biosynthesis of allophycocyanin holo-α subunits (named as holo-ApcAS for Synechocystis sp. PCC 6803 and holo-ApcAT for T. elongatus BP-1) in Escherichia coli. The two holo-ApcAs were successfully reconstituted in E. coli and purified by metal affinity chromatography. Spectral analysis showed that the two proteins have similar spectroscopic properties, with absorbance maximum both at 614nm and emission maximum at 639nm for holo-ApcAS and 638nm for holo-ApcAT. At high temperature, the recombinant holo-ApcAT was much more stable than the recombinant holo-ApcAS. Holo-ApcAS was most fluorescent at pH 8.5 and stable in pH range of 6.0-9.0 while holo-ApcAT was most fluorescent at pH 6.0 and stable in pH range of 5.0-7.0, with residual fluorescence intensity no less than 90% of the maximum fluorescence. These findings will pave the way for further protein engineering to achieve high stable APC from extremophiles.
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China. Electronic address: chenhx001@126.com.
authors.forename: Huaxin
authors.initials: H
authors.lastname: Chen
authors.affiliation: Qingdao University of Science and Technology, Qingdao 266071, China.
authors.forename: Qiuzi
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China. Electronic address: Jiangpeng@qdio.ac.cn.
authors.forename: Peng
authors.initials: P
authors.lastname: Jiang
mesh_terms: D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D015870:Gene Expression; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010798:Phycocyanin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D055550:Protein Stability; D021122:Protein Subunits / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D046939:Synechocystis / Q000737:chemistry*
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; C002497:allophycocyanin; D010798:Phycocyanin
keywords: Allophycocyanin; Escherichia coli; Fluorescence; Thermostability