Identification and characterization of partner proteins interacting with fatty acid activation enzyme Slr1609 in Synechocystis sp. PCC 6803 / 生物工程学报

ABSTRACT

To understand molecular modules related to polyunsaturated fatty acids (PUFA) synthesis and eventually produce PUFA at high efficiency, we developed a protein complex analysis technology in Synechocystis sp. PCC 6803, and applied it to identify possible partner proteins interacting with the key enzymes that catalyze PUFA biosynthesis. We first constructed a recombinant expression of protein of slr1609 encoding the fatty acid activation enzyme, by fusing 3xFLAG tag with the target protein. Then we verified its expression by Western blotting targeting 3xFLAG tag. To maximize purification of Slr1609 protein complex, we optimized the protein expression conditions of Slr1609 in Synechocystis in a 5 L fermenter by monitoring its gene expression using RT-qPCR. The purification of the Slr1609 protein complexes was demonstrated by a Native-PAGE analysis. Finally, LC-MS/MS proteomic analysis allowed identification of the possible partner proteins interacting with Slr1609.