藻红蓝蛋白β亚基体内重组及其色谱分析
CHROMATOGRAPHY ANALYSIS OF PEPTIDES FROM THE B-SUBUNIT OF PHYCOERYTHROCYANIN IN VIVO RECONSTITUTION
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摘要: 藻胆蛋白是蓝藻中的捕光蛋白,其生物合成的重要一步是藻胆色素与脱辅基蛋白的连接。大多数藻胆色素的正确连接都需要结合位点专一和对色素的构象有选择性的裂合酶来催化完成,但是这方面的报道不是很多。藻红蓝蛋白由两个亚基组成,β亚基(简称β-PEC)含171个氨基酸残基及两个辅基色素藻蓝胆素(简称PCB),分别在Cys-84和Cys-155位以硫醚键共价相连。通过同源性分析获得的由编号为alr0617基因编码的蛋白为藻红蓝蛋白β亚基(β-PEC)中的Cys-84与PCB的连接的催化酶。为了研究层理鞭枝藻藻红蓝蛋白(PEC)β亚基(β-PEC)中藻蓝胆素(PCB)与脱辅基蛋白的连接机制,通过体内重组方式得到色素蛋白PCB-PecB(C155I),分析表明该色素蛋白与β-PEC的吸收光谱和荧光光谱一致。酸性尿素变性实验证明得到的色素蛋白中的藻蓝胆素PCB没有被破坏。使用胃蛋白酶对天然藻红蓝色素蛋白和重组藻红蓝色素蛋白进行相同条件的水解并得到各自的色素肽,高效液相色谱分析表明这两种色素肽相同,由此证明了编号为alr0617基因编码的蛋白质能催化PCB与PecB(C155I)正确共价偶联。Abstract: Phycobiliproteins are ligh-t harvesting proteins present in cyanobacteria. The most important step in phycobilin biosynthesisis the phycobilin addition to the apophycobiliproteins. In vivo, the correct attachment of most chromophores is catalyzed bybinding-site and chromophore-specific lyases, of which only few have hitherto been characterized. Phycoerythrocyanin have twosubunits. Bsubunit of phycoerythrocyanin is made of 171 amino acids and the binding-sites of phycobilin addition to the apophycobiliproteinsare Cys- 84 and Cys-155. The protein encoded by gene alr0617 was proved to be the lyase of Cys-84 of B- PEC. Bsubunit of phycoerythrocyanin ( PCB-PecB( C155I) ) was obtained by the in vivo reconstitution. The veracity was shown throughthe absorption spectra and the fluorescence spectra. After denaturation in the dark, with urea in the presence of hydrochloric acid( pH= 2), the phycobilin ( PCB) was not destroyed. The phycocyanobilin peptides were obtained from the natural subunit ofphycoerythrocyanin( B- PEC) and reconstituted PCB-PecB( C155I)hydrolyzed by pepsin, respectively. With Chromatography ana-lysis of high pressure liquid chromatography, we make a comparison of the retention time between the natural and the reconstitutedpept ides. The results showed that genes for the lyase ( alr 0617) catalyzed PCB attachment sites at Cys-84 in B-PEC.
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Keywords:
- B-PEC /
- Reconstitution in vivo /
- Hydrolysis /
- Chromatography analysis
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