蓝藻红色荧光蛋白All1280 GAF2在E. coli BL21(DE3)中的表达及其突变体构建（英文）
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摘要
采用PCR技术从鱼腥藻(Anabaena sp.) PCC 7120中扩增获得红色荧光蛋白基因all1280 gaf2,并利用Bam HⅠ和SalⅠ酶切位点,将该基因插入到pET-30a(+)中,构建表达载体pET-all1280 gaf2。将该表达载体与藻胆色素生物合成质粒pACYC-ho1-pcyA同时转化到大肠杆菌E. coli BL21 (DE3),表达后获得大肠杆菌色素细胞。结果显示,该色素细胞在荧光显微镜下具有红色荧光,且在15E/15Z态之间具有可逆光效应。进一步以pET-all1280 gaf2为模板,通过定点突变技术在all1280 gaf2基因中引入C53A突变,获得了突变体All1280 GAF2 (C53A)。将All1280 GAF2 (C53A)与藻胆色素在E. coli BL21 (DE3)中共表达,获得了比野生型红色荧光更强的大肠杆菌色素细胞。研究结果表明,与野生型相比,All1280 GAF2 (C53A)具有较高的摩尔消光系数和荧光量子产率,红色荧光更强。
Cyanobacteriochromes contain GAF( cGMP phosphodiesterase,adenylyl cyclase,and Fhl A protein) domains in the N-terminal region that bind phycocyanobilin autocatalytically.In the current study,we amplified the cyanobacteriochrome gene fragment of all1280 gaf2 from Anabaena sp. PCC 7120 using PCR,and then inserted it into pET-30 a( +). For over-expression,both pET-all1280 gaf2 and pACYC-ho1-pcyA,which catalyze phycocyanobilin( PCB) biosynthesis,were transformed into E. coli BL21( DE3). Cells harboring pET-all1280 gaf2 and pACYC-ho1-pcyA expressed chromophorylated All1280 GAF2 successfully. Results showed that All1280 GAF2 underwent reversible photoconversion between the 15 E form( λmax= 560 nm) and 15 Z form( λmax= 413 nm). Using fluorescent microscopy,we detected a red fluorescence/no fluorescence reversible photoconversion of All1280 GAF2 in E. coli BL21( DE3). Cys53 was essential for photoconversion of All1280 GAF2 because its mutagenesis resulted in a PCB adduct,which exhibited no photoconversion but stable red fluorescence.Compared with the wild-type,All1280 GAF2( C53 A) had stronger red fluorescence with higher extinction coefficients and fluorescence yields. It is expected that these two constructs could serve well in the labeling of living cells.
Cyanobacteriochromes contain GAF( cGMP phosphodiesterase,adenylyl cyclase,and Fhl A protein) domains in the N-terminal region that bind phycocyanobilin autocatalytically.In the current study,we amplified the cyanobacteriochrome gene fragment of all1280 gaf2 from Anabaena sp. PCC 7120 using PCR,and then inserted it into pET-30 a( +). For over-expression,both pET-all1280 gaf2 and pACYC-ho1-pcyA,which catalyze phycocyanobilin( PCB) biosynthesis,were transformed into E. coli BL21( DE3). Cells harboring pET-all1280 gaf2 and pACYC-ho1-pcyA expressed chromophorylated All1280 GAF2 successfully. Results showed that All1280 GAF2 underwent reversible photoconversion between the 15 E form( λmax= 560 nm) and 15 Z form( λmax= 413 nm). Using fluorescent microscopy,we detected a red fluorescence/no fluorescence reversible photoconversion of All1280 GAF2 in E. coli BL21( DE3). Cys53 was essential for photoconversion of All1280 GAF2 because its mutagenesis resulted in a PCB adduct,which exhibited no photoconversion but stable red fluorescence.Compared with the wild-type,All1280 GAF2( C53 A) had stronger red fluorescence with higher extinction coefficients and fluorescence yields. It is expected that these two constructs could serve well in the labeling of living cells.
引文
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[13]Song JY,Cho HS,Cho JI,Jeon JS,Lagarias JC,et al.Near-UV cyanobacteriochrome signaling system elicits negative phototaxis in the cyanobacterium Synechocystis sp.PCC 6803[J].Proc Natl Acad Sci USA,2011,108(26):10780-10785.
[14]Narikawa R,Enomoto G,Win NN,Fushimi K,Ikeuchi M.A new type of dual-cys cyanobacteriochrome GAF domain found in cyanobacterium Acaryochloris marina,which has an unusual red/blue reversible photoconversion cycle[J].Biochem,2014,53(31):5051-5059.
[15]Rodriguez EA,Campbell RE,Lin JY,Lin MZ,Miyawaki A,et al.The growing and glowing toolbox of fluorescent and photoactive proteins[J].Cell,2017,42(2):111-129.
[2]Fushimi K,Nakajima T,Aono Y,Yamamoto T,Win NN,et al.Photoconversion and fluorescence properties of a red/green-type cyanobacteriochrome AM1_C0023g2 that binds not only phycocyanobilin but also biliverdin[J].Front Microbiol,2016,26(7):1-12.
[3]Rei N,Takahiro N,Yuki A,Keiji F,Gen E,et al.A biliverdin-binding cyanobacteriochrome from the chlorophyll dbearing cyanobacterium Acaryochloris marina[J].Sci Rep,2015,7950(5):1-10.
[4]Banerjee S,Schenkelberg CD,Jordan TB,Reimertz JM,Crone EE,et al.Mispacking and the fitness landscape of the green fluorescent protein chromophore milieu[J].Biochem,2017,56(5):736-747.
[5]Hansen S,Stüber JC,Ernst P,Koch A,Bojar D,et al.Design and applications of a clamp for green fluorescent protein with picomolar affinity[J].Sci Rep,2017,7:1-14.
[6]Sun YF,Xu JG,Tang K,Dan M,Grtner W,et al.Orange fluorescent proteins constructed from cyanobacteriochromes chromophorylated with phycoerythrobilin[J].Photochem Photobiol,2014,13(5):757-763.
[7]Ma Q,Zhou Z,Dong JZ,Zhou M.Molecular structure,biosynthesis and photoreversibility of cyanobacteriochromes[J].Plant Sci J,2014,32(5):522-530.
[8]Ulijasz AT,Cornilescu G,Stetten D,Cornilescu C,Velazquez EF,et al.Adaptive and acclimative responses of cyanobacteria to far-red light[J].Environ Microbiol,2015,17(10):3450-3465.
[9]Zhang J,Wu XJ,Wang ZB,Chen Y,Wang X,et al.Fused-gene approach to photoswitchable and fluorescent biliproteins[J].Angew Chem Int Ed Engl,2010,49:5456-5458.
[10]Chen Y,Zhang J,Luo J,Tu JM,Zeng XL,et al.Photophysical diversity of two novel cyanobacteriochromes with phycocyanobilin chromophores:photochemistry and dark reversion kinetics[J].FEBS J,2012,279:40-54.
[11]Pennacchietti F,Losi A,Xu XL,Zhao KH,Grtner WG,et al.Photochromic conversion in a red/green cyanobacteriochrome from Synechocystis PCC6803:quantum yields in solution and photoswitching dynamics in living E.coli cells[J].Photoch Photobio Sci,2015,2:229-237.
[12]Reichhart E,Ingles-Prieto A,Tichy AM,McKenzie C,Janovjak H.A phytochrome sensory domain permits receptor activation by red light[J].Angew Chem Int Ed Engl,2016,55(21):6339-6342.
[13]Song JY,Cho HS,Cho JI,Jeon JS,Lagarias JC,et al.Near-UV cyanobacteriochrome signaling system elicits negative phototaxis in the cyanobacterium Synechocystis sp.PCC 6803[J].Proc Natl Acad Sci USA,2011,108(26):10780-10785.
[14]Narikawa R,Enomoto G,Win NN,Fushimi K,Ikeuchi M.A new type of dual-cys cyanobacteriochrome GAF domain found in cyanobacterium Acaryochloris marina,which has an unusual red/blue reversible photoconversion cycle[J].Biochem,2014,53(31):5051-5059.
[15]Rodriguez EA,Campbell RE,Lin JY,Lin MZ,Miyawaki A,et al.The growing and glowing toolbox of fluorescent and photoactive proteins[J].Cell,2017,42(2):111-129.