嗜热藻BP-1中几个蓝细菌光敏色素结构域体内重组及光化学性质研究

刘冰冰; 陈煜; 邓明刚; 赵开弘; 周明

doi:10.3724/SP.J.1142.2013.20178

嗜热藻BP-1中几个蓝细菌光敏色素结构域体内重组及光化学性质研究

1.
华中农业大学生命科学技术学院, 武汉 430070
2. 华中科技大学环境科学与工程学院, 武汉 430074
3. 华中农业大学农业微生物学国家重点实验室, 武汉 430070

基金项目: 国家自然科学基金资助项目(21072068; 31110103912 )。

详细信息

作者简介:
刘冰冰(1987-),女,河南三门峡人,硕士研究生,主要从事蓝细菌光合作用和蛋白质工程研究。

通讯作者:
周明, E-mail: zhouming321@126.com

中图分类号: Q71
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出版历程
- 收稿日期: 2012-07-05
- 修回日期: 2012-11-18
- 发布日期: 2013-04-29

Reconstitution in vivo and Photochemical Properties of Several Cyanobacteriochrome Domains from Thermosynechococcus elongatus BP-1

1.
College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
2. College of Environment Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, China
3. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China

摘要

摘要: 通过蛋白质序列同源性比对分析,在嗜热藻(Thermosynechococcus elongatus BP-1)里面找到了与已知的Pb/Pg型蓝细菌光敏色素TePixJ和TeTlr0924同源的3个基因tlr0911、 tlr1215和tlr1999。通过分子克隆技术把它们的GAF结构域分别构建在pET30a(+)表达载体上,与可生成藻蓝胆素(PCB)的质粒pACYCDuet-ho1-pcyA在大肠杆菌BL21(DE3)体内重组,生成重组蛋白,利用亲和层析柱分离纯化,纯化后的蛋白质经过锌荧光和蛋白质酸性尿素变性以及荧光光谱和吸收光谱等实验分析鉴定,结果表明,Tlr0911-GAF存在蓝光吸收态Pb_{406 nm}和绿光吸收态Pg_{527 nm}之间的可逆光转换,它可共价结合两种藻胆色素,即藻紫胆素(PVB)和藻蓝胆素(PCB),Tlr1999-GAF则存在蓝光吸收态Pb_{417 nm}和青光吸收态Pt_{496 nm}之间的可逆光转换,它同样共价结合PVB和PCB, 而Tlr1215-GAF1和Tlr1215-GAF2不能自发结合藻胆色素,不具有光活性。
- 蓝细菌光敏色素 /
- 可逆光转换 /
- 体内重组
Abstract: By protein sequence homology comparison with the Pb/Pg-type cyanobacteriochrome TePixJ and TeTlr0924, three homologous genes tlr0911, tlr1215 and tlr1999 from Thermosynechococcus elongatus BP-1 were found. By molecular cloning techniques, the GAF domains of those genes were cloned into expression vector pET30a(+), respectively. The E.coli strain BL21 (DE3) harboring the expression plasmid and the pACYCDuet-ho1-pcyA plasmid for phycocyanobilin (PCB) were induced to generate recombinant proteins. The expressed proteins (His)₆ tagged at the N-terminus were purified with nickel-affinity His-Trap chelating column. The purified proteins were identified with zinc-induced fluorescence, acidic urea denaturation, fluorescence and absorption spectrum. Results showed that Tlr0911-GAF contained two covalently bound bilin chromophores, phycoviolobilin (PVB) and phycocyanobilin (PCB), and exhibited reversible photoconversion between a blue-absorbing form at 406 nm (Pb_{406 nm}) and a green-absorbing form at 527 nm (Pg_{527 nm}). Tlr1999-GAF was also covalently bound with PVB and PCB, and reversible photoconversion existed between a blue-absorbing form at 417 nm (Pb_{417 nm}) and a teal-absorbing form at 496 nm (Pt_{496 nm}). Neither Tlr1215-GAF1 nor Tlr1215-GAF2 could be spontaneously bound with bilin chromophore.
- Cyanobacteriochrome /
- Reversible photoconversion /
- Assembly in Escherichia coli

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参考文献(14)

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