| Development of genetic engineering and synthetic biology has made it possibleto produce biofuels and chemicals in photosynthetic cyanobacteria that utilize solarenergy and CO2directly as the sole energy and carbon sources, respectively. However,current productivity in cyanobacterial systems remains at level far lower than theother engineering hosts, which may be due to the fact that information related toenzymes in important biosynthesis pathways is limited in cyanobacteria, and this hassignificantly restricted their application potentials. To address the issue, methods areneeded to obtain and characterize proteins from cyanobacteria cells. Fusion tagtechnology that has been successfully applied in many microorganisms, offers a goodoption to achieve this goal in cyanobacteria. However, due to the abundant membranesystems and the relatively low protein expression levels in cyanobacteria, it ischallenging to develop and optimize the fusion tag method for cyanobacteria.To establish a method of separating and purifying protein in cyanobacteria cells,the model cyanobacteria Synechocystis sp. PCC6803was chosen as experimentobject, and the membrane protein Δ-6acyl-lipid desaturase and Δ-9acyl-lipiddesaturase as the isolation targets. Mutants were constructed in that the target proteinswere coupled with His tag or3×FLAG tag by employing fusion PCR and homologousintegration. The expression of the target prteins was optimized by qRT-PCR, and theirsubcellular location was determined by Western blotting after ultracentrifugation andsolubilizing biological membrane, and scaled up cell cultivation in5-L fermenters. Inaddition, attempts were made to separate and purify possible protein complexassociated with Δ-9acyl-lipid desaturase proten.In this study, the methods of separating and purifying target proteins wasdeveloped and optimized for cyanobacteria, which constitutes an importantfoundation for further characterizing key enzymes related to important biosynthesispathways in cyanobacteria. |
PDF Full Text Request