蓝细菌光敏色素Alr1966GAF2及其突变体的表达与光化学性质研究

马琼; 向建伟; 牟若兰; 邓腊青; 王运; 周明

doi:10.11913/PSJ.2095-0837.2020.40551

蓝细菌光敏色素Alr1966GAF2及其突变体的表达与光化学性质研究

马琼^1,2, ,,
向建伟^1,2,
牟若兰^1,2,
邓腊青^1,2,
王运^1,2,
周明³

1. 湖北民族大学生物资源保护与利用湖北省重点实验室, 湖北恩施 445000;
2. 湖北民族大学生物科学与技术学院, 湖北恩施 445000;
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:

恩施州科技计划项目（D20190025）；国家自然科学基金（21472055）；湖北民族大学高水平科研成果校内培育项目（PY20020）；湖北民族大学大学生科研训练项目（X201910517274， X201910517231）。

详细信息

作者简介:
马琼(1979-),女,博士,副教授,研究方向为蓝细菌光合作用与蛋白质工程(E-mail:maqiong110@126.com)。

通讯作者:
马琼,E-mail:maqiong110@126.com

中图分类号: Q946
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出版历程
- 收稿日期: 2019-10-23
- 修回日期: 2019-12-25
- 网络出版日期: 2022-10-31
- 发布日期: 2020-08-27

Expression and photochemical properties of cyanobacteriochrome Alr1966GAF2 and its mutants

1. Key Laboratory of Biologic Resources Protection and Utilization of Hubei Province, Hubei University for Nationalities, Enshi, Hubei 445000, China;
2. College of Biological Science and Technology, Hubei University for Nationalities, Enshi, Hubei 445000, China;
3. Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China

Funds:

This work was supported by grants from the Project of Science and Technology of Enshi (D20190025), National Natural Science Foundation of China (21472055), Incubation Project for High-Level Scientific Research Achievements of Hubei Minzu University(PY20020), and Project of Student Research Training from Hubei Minzu University (X201910517274, X201910517231).

摘要

摘要: 采用PCR技术从鱼腥藻（Anabaena sp.PCC7120）中扩增蓝细菌光敏色素基因片段alr1966gaf2，将alr1966gaf2插入到pET-30a（+）载体中，构建表达质粒pET-alr1966gaf2。最后将Alr1966GAF2与HO1、PcyA在E.coli BL21（DE3）中共表达获得色素蛋白Alr1966GAF2，并对该蛋白的光化学性质进行分析。结果显示，色素蛋白Alr1966GAF2结合色素为藻蓝胆素（phycoerythrobilin，PCB）或藻紫胆素（phycoviolobilin，PVB），在3种不同吸收态15Z-P_{428 nm}、中间态和15E-P_{514 nm}之间具有顺序可逆光效应。通过定点突变技术将DXCF基序中的保守性Cys突变为Ala，获得了突变体Alr1966GAF2（C72A）。将Alr1966GAF2（C72A）与HO1、PcyA共表达，获得色素蛋白Alr1966GAF2（C72A）。研究结果表明Alr1966GAF2（C72A）结合色素为PCB，Alr1966GAF2（C72A）-PCB具有较强的荧光活性，其荧光量子的产率高达0.11。Alr1966GAF2（C72A）不仅能够共价结合PCB，还可以结合胆绿素（Biliverdin，BV），均具有较强的红色荧光活性。
- 蓝细菌光敏色素 /
- 顺序可逆光效应 /
- 红色荧光蛋白 /
- 胆绿素
Abstract: To construct pET-alr1966gaf2, a fragment of alr1966gaf2 was amplified by polymerase chain reaction (PCR) from Anabaena sp. PCC7120, and then inserted into pET-30a(+). For over-expression, pET-alr1966gaf2 was transformed into Escherichia coli BL21(DE3) containing pACYC-ho1-pcyA and biliproteins were co-expressed successfully. Results showed that bili-Alr1966GAF2 had a sequential reversible photoconversion in three different states. We also detected red fluorescence reversible photoconversion of Alr1966GAF2 in 15E-P_{514 nm}/15Z-P_{428 nm} forms. Via site-directed mutagenesis, we mutated conserved Cys into Ala in the conserved DXCF motif of Alr1966GAF2, resulting in Alr1966GAF2(C72A). Alr1966GAF2(C72A)-PCB showed strong red fluorescence and high fluorescence quantum yield of 0.11. Furthermore, Alr1966GAF2(C72A) could bind to phycoerythrobilin (PCB) and biliverdin (BV) covalently, with strong red fluorescence. Therefore, as a red fluorescent protein, Alr1966GAF2(C72A) could be further developed as a fluorescent probe and applied in life sciences.
- Cyanobacteriochrome /
- Sequential reversibility /
- Red fluorescent protein /
- Biliverdin

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参考文献(16)

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