藻蓝蛋白和藻红蓝蛋白β亚基半胱氨酸的定点突变及体外重组研究
SITE-DIRECTED MUTATION OF THE β-SUBUNIT OF PHYCOCYANIN AND PHYCOERYTHROCYANIN AND THE STUDY OF RECONSTITUTION IN VITRO
-
摘要: 为研究藻蓝蛋白(PC)和藻红蓝蛋白(PEC)β亚基(分别简称为β-PC、β-PEC)生物合成、结构与功能的关系,用Mega primer PCR定点突变技术设计β-PC、β-PEC中与藻蓝胆素连接的第二个半胱氨酸的定点突变蛋白质β-PC(C1I)和β-PEC(C1I).将相应的基因片段亚克隆于表达载体pET 30a,并转化大肠杆菌BL2l(DE3).经IPTG诱导后,β-PC(C1I)和β-PEC(C1I)在大肠杆菌中均得到了高效表达.β-PC(C1I)和β-PEC(C1I)与藻蓝胆素的重组结果表明两个突变体的结构基本没发生改变,有利于对β-PC和β-PEC的生物合成进行进一步研究.Abstract: To study the structure-function relationships of the β subunit of phycocyanin(β-PC)and phycoerythrocyanin(β-PEC),site-directed mutation was used to generate two mutants,β-PC(C155I)and β-PEC(C155I). The six primers were designed,of which primer P1,P2 and P3 were used to generate mutant β-PC(C155I),primer P4,P5 and P6 were used to generate β-PEC(C155I). Both of primer P3 and P6 comained a restriction site EcoRⅤ, so we can pick out the cysteine mutants from the reconstructed plasmids.The PCR products were purified and digested with Sma Ⅰ and XhoⅠ, then recombined with pBluescript vector and transfonned into the E.coli TG1 strain. It was confirmed that the genes were inserted into pBluescript vector by the restriction enzyme double digestion with Sma Ⅰ and Xho Ⅰ,Sma Ⅰ and EcoRⅤ,PCR and sequencing.The genes of the two cysteine mutants were purified after the Sma Ⅰ/Xho Ⅰ double digestion of the reconstructed plasmids,and then inserted into expression vector pET 30a,finally transformed into E. coli BL21(DE3).In the transformed BL21(DE3)strains,24kD protein were overexpressed,respectively.The overexpressed proteins were direct used to reconstitution with pigment PCB in vitro.The purified reconstituted products were determined with absorption spectra and fluorescence spectra,showing that they were similar to the β-PC and β-PEC in spectrum characteristics. It was analysed by GorⅣtoo, showing that there was no other change happened in the secondary structure of β-PC and its mutant,except that Cys-155 of β-PC was situated at the C-terminal of random coil of ANDPNGITKGDC amino acid residues,whereas at N-terminal of alpha helix of ISALISEVASYFDRA in β-PC(C155I).And the similar situation happened to β-PEC and its mutant.So the structure of the two mutants was similar to the wild-type proteins.
-
-
[1] Foerstendorf H,Parbel A,Scheer H,et al.E isomerization of the α-84 phycoviolobilin chromophore of phycoerythrocyanin from Mastigocladus laminosus investigated by Fourier-transform infrared difference spectroscopy [J].FEBS Letters,1997,402:173-176[2] MacColl Robert,Eisele Leslie E,Marrone Jennifer,Fluorescence polarization studies on four biliproteins and a bilin model for phycoerythrin [J].Biochimica et Biophysica Acta,1999,1412:230-239[3] Zhao K H,Deng M G,Zheng Min,Novel activity of a phycobiliprotein lyase:both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon[J].FEBS Letters, 2000,469:9-13[4] MacColl Robert,Cyanobacterial Phycobilisomes [J].Journal of Structural Biology,1998,124:311-334[5] Zhang J M,Zhao J Q,Jiang L J,Studies on the energy transfer among the rod-core complex from phycobilisome of Anabaena variabilis by time resolved fluorescence emission and anisotropy spectra[J].Biochimica et Biophysica Acta,1997,1320:285-296[6] Ausubel F M,Brent R,Kingston R E,et al.Short Protocols in Molecular Biology[M].John Wiley Sons,Inc,1995,268-271[7] Andrey A,Demidov and Mamoru Mimuro,Deconvolution of C-Phycocyanin β-84 and β-155 Chromophore Absorption and Fluorescence Spectra of Cyanobacterium Mastigocladus laminosus [J].Biophysical Journal,1995,68:1500-1506[8] Axel Parbel,Zhao K H,Jacqus Breton,et al.Chromophore assignment in phycoerythrocyanin from Mastigocladus laminosus[J].Photosynthesis reseach,1997,54:25-34[9] Duerring M,Huber R,Bode W,et al.Refined three-dimensional structure of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus at 2.7A[J].J.Mol Biol, 1990,211(3):633-644[10] Sheng S B,Zhang F T,Deng M G,et al.Clonging and overexpressing of phycoerythrocyanin operon E-gene of Mastigocladus Laminosus in Escherichia coli [J].Acta Hydrobioloica Sinica, 2001,25(4):427-430.[盛树斌,张富铁,邓明刚,等.层理鞭枝藻藻红蓝蛋白E基因片段的克隆和表达.水生生物学报,2001,25(4):427-430][11] Ma C,Zhou M,Zhang F T,et al.Cloning,Expressing and Activities Study of Mastigocladus laminosus PecE Mutated Genes [J].Acta Hydrobioloica Sinica,2003,27(6):614-618.[马聪,周明,张富铁等.藻红蓝蛋白裂合异构酶E基因突变体的构建、表达与酶活性检测.水生生物学报,2003,27(6):614-618][12] Zheng M,Da L,Deng M G,et al.Clonging and overexpressing of phycoerythrocyanin operon F-gene of Mastigocladus Laminosus in Escherichia coli[J].Acta Hydrobioloica Sinica,2002,26(2):168-174.[郑敏,答亮,邓明刚,等.层理鞭枝藻藻红蓝蛋白F基因片段的克隆和表达.水生生物学报,2002,26(2):168-174] Foerstendorf H,Parbel A,Scheer H,et al.E isomerization of the α-84 phycoviolobilin chromophore of phycoerythrocyanin from Mastigocladus laminosus investigated by Fourier-transform infrared difference spectroscopy [J].FEBS Letters,1997,402:173-176[2] MacColl Robert,Eisele Leslie E,Marrone Jennifer,Fluorescence polarization studies on four biliproteins and a bilin model for phycoerythrin [J].Biochimica et Biophysica Acta,1999,1412:230-239[3] Zhao K H,Deng M G,Zheng Min,Novel activity of a phycobiliprotein lyase:both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon[J].FEBS Letters, 2000,469:9-13[4] MacColl Robert,Cyanobacterial Phycobilisomes [J].Journal of Structural Biology,1998,124:311-334[5] Zhang J M,Zhao J Q,Jiang L J,Studies on the energy transfer among the rod-core complex from phycobilisome of Anabaena variabilis by time resolved fluorescence emission and anisotropy spectra[J].Biochimica et Biophysica Acta,1997,1320:285-296[6] Ausubel F M,Brent R,Kingston R E,et al.Short Protocols in Molecular Biology[M].John Wiley Sons,Inc,1995,268-271[7] Andrey A,Demidov and Mamoru Mimuro,Deconvolution of C-Phycocyanin β-84 and β-155 Chromophore Absorption and Fluorescence Spectra of Cyanobacterium Mastigocladus laminosus [J].Biophysical Journal,1995,68:1500-1506[8] Axel Parbel,Zhao K H,Jacqus Breton,et al.Chromophore assignment in phycoerythrocyanin from Mastigocladus laminosus[J].Photosynthesis reseach,1997,54:25-34[9] Duerring M,Huber R,Bode W,et al.Refined three-dimensional structure of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus at 2.7A[J].J.Mol Biol, 1990,211(3):633-644[10] Sheng S B,Zhang F T,Deng M G,et al.Clonging and overexpressing of phycoerythrocyanin operon E-gene of Mastigocladus Laminosus in Escherichia coli [J].Acta Hydrobioloica Sinica, 2001,25(4):427-430.[盛树斌,张富铁,邓明刚,等.层理鞭枝藻藻红蓝蛋白E基因片段的克隆和表达.水生生物学报,2001,25(4):427-430][11] Ma C,Zhou M,Zhang F T,et al.Cloning,Expressing and Activities Study of Mastigocladus laminosus PecE Mutated Genes [J].Acta Hydrobioloica Sinica,2003,27(6):614-618.[马聪,周明,张富铁等.藻红蓝蛋白裂合异构酶E基因突变体的构建、表达与酶活性检测.水生生物学报,2003,27(6):614-618][12] Zheng M,Da L,Deng M G,et al.Clonging and overexpressing of phycoerythrocyanin operon F-gene of Mastigocladus Laminosus in Escherichia coli[J].Acta Hydrobioloica Sinica,2002,26(2):168-174.[郑敏,答亮,邓明刚,等.层理鞭枝藻藻红蓝蛋白F基因片段的克隆和表达.水生生物学报,2002,26(2):168-174]
计量
- 文章访问数: 927
- HTML全文浏览量: 3
- PDF下载量: 444
下载: