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Protein Complexes Related To Polyunsaturated Fatty Acid Biosynthesis In Synechocystis Sp. PCC6803

Posted on:2015-01-02Degree:MasterType:Thesis
Country:ChinaCandidate:Q WuFull Text:PDF
GTID:2180330452969924Subject:Bio-engineering
Abstract/Summary:PDF Full Text Request
Metabolic activities in cells are conducted by protein complexs that areimportant in a variety of life processes, such as biocatalysis, transport, signaltransduction, immunologic process and cell regulation. Currently, little knowledge isavailable on how proteins work in cyanobacterial metabolic and regulatory pathways,which has restricted various attempts to increase the yield of polyunsaturated fattyacids (PUFA)—a kind of high-value-added bio-products. To improve understandingof biological modules related to PUFA production and eventually produce PUFA athigh efficiency, we aimed to develop a protein complex analysis technology inSynechocystis sp. PCC6803, and apply it to identify partner proteins of the keyenzymes that catalyze PUFA biosynthesis. Our research hypothesis is that the proteinsassociated with the key PUFA biosynthetic enzymes in the same protein complex mayalso be important for PUFA biosynthesis.In this study, we selected three genes related to PUFA biosynthesis inSynechocystis sp. PCC6803: slr1609, sll0617, slr1137. The recombinant protein wasgenerated by fusing3×FLAG tag with the target protein and the mutants were verifiedby sequencing. Then, we chose the mutant strain with recombination of slr1609genewhich encodes fatty acid activation enzyme for further analysis. The expressioncondition of the recombinant protein was optimized by RT-qPCR and the3×FLAGtag was identified by SDS-PAGE and Western blotting. According to the subcellularlocalization of Slr1609and the principle of affinity purification, both the targetprotein complexes in the whole cell and in cytoplasm were extracted and purified, andthe existence of protein complexes in the purified protein mixture was confirmed bynative-PAGE. Finnaly, we determined the possible partner proteins of Slr1609byLC-MS/MS proteomic analysis. Although further confirmation is still needed, theanalysis allowed identification of three hypothetical proteins, Sll0350, Ssl1707,Sll0981, as possible partner proteins ralated to fatty acid metabolism.The major aim of the study was to develope the protein complex analysistechnology, and apply it to explore fatty acid synthesis in cyanobacteria. The study ofthe fatty acid metabolic pathway is important for productivity improvement incyanobacterial systems. In addition, the method of protein complex analysisdeveloped here, once established, can be used in other microbial systems as well.
Keywords/Search Tags:Synechocystis sp. PCC6803, fatty acid, protein complexes, proteomics
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