Biosynthesis, spectral properties and thermostability of cyanobacterial allophycocyanin holo-α subunits

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• 作者：Huaxin Chen^a ; ^b ; ^{chenhx001@126.com" class="auth_mail" title="E-mail the corresponding author} ; Qiuzi Liu^c ; Jin Zhao^a ; ^b ; Peng Jiang^a ; ^b ; ^{Jiangpeng@qdio.ac.cn" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Allophycocyanin ; Escherichia coli ; Thermostability ; Fluorescence
• 刊名：International Journal of Biological Macromolecules
• 出版年：2016
• 出版时间：July 2016
• 年：2016
• 卷：88
• 期：Complete
• 页码：88-92
• 全文大小：626 K

文摘

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 614 nm and emission maximum at 639 nm for holo-ApcAS and 638 nm 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.