单色素藻胆蛋白亚基的蛋白质工程研究-环境科学专业论文.docx

华中科技 大学硕士 学 华 中 科 技 大 学 硕 士 学 位 论 文 II II Abstract Phycobilisomes serve as the light-harvesting antenna in cyanobacteria and red algae. These supramolecular protein complexes, are primarily composed of phycobiliproteins, a brilliantly colored family of proteins bearing covalently attached, open-chain tetrapyrroles known as phycobilins. In additional, phycobilisomes also contain smaller amounts of linker peptides, which are required for proper assembly and functional organization of phycobilisomes. Phycobilin chromophores are generally bound to apoprotein at conserved positions by cysteinyl thioether linkages to generate phycobiliproteins. Based on their absorption spectral properties, phycobiliproteins have been assigned to four classes: phycocyanin (PC), phycoerythrin (PE), allophycocyanin (APC) and phycoerythrocyanin (PEC), in which the latter is primarily found in certain filamentous, heterocyst-forming cyanobacteria. In vivo, the correct attachment of most chromophores is catalyzed by lyases, of which only few have hitherto been characterized. As we know now, PecA and PecF catalyzed both the attachment and isomerization of phycocyanobilin to PecA. The first step in the pathway of bilin biosynthesis is the conversion of heme to biliverdin IX (BVD) by the heme oxygenase encoded by ho1, then PCB:ferredoxin oxidoreductase encoded by pcyA catalyze BVD to 3Z-PCB. By means of molecular biology technique, pETDuet-pecA had been obtained. Then pecA and pecE/pecF was introduced into E. coli, respectively, together with genes hol and pcyA. PecA, PecE/PecF, HO1 and PcyA were overexpressed in E. coli, and PecE/PecF could isomerize PCB to PVB and attach PVB to PecA. It was demonstrated by spectral analyses and the classical phototransformation of ?-PEC. DNA segments were obtained by Bsp1431 partial digestion on genomic DNA of Synechocystis sp. strain PCC 6803. DNA segments were introduced into target carrier pTRG and the genomic DNA library was set up. The protein interaction