The photosynthetic cyanobacterium Synechocystis PCC 6803, with a known prokaryotic genome was investigated for assessing the expression of an animal protein and as a candidate for future biotechnological investigations and manipulations. Owing to the relatively simple genetic structure, its own transformable ability and complete sequenced genome, the cyanobacterium Synechocystis PCC 6803 became a good model system to study the expression of a single chain antibody, DB3 V_HR100/K. In this study, I demonstrated the transformation frequency by electroporation. A proper insertion site of the rfbJ gene was investigated. By random insertional mutagenesis, some native strong promoters for foreign gene expression were found and examined. Several mutant strains expressing DB3 V_HR100/K were establ...[ Read more ]

The photosynthetic cyanobacterium Synechocystis PCC 6803, with a known prokaryotic genome was investigated for assessing the expression of an animal protein and as a candidate for future biotechnological investigations and manipulations. Owing to the relatively simple genetic structure, its own transformable ability and complete sequenced genome, the cyanobacterium Synechocystis PCC 6803 became a good model system to study the expression of a single chain antibody, DB3 V_HR100/K. In this study, I demonstrated the transformation frequency by electroporation. A proper insertion site of the rfbJ gene was investigated. By random insertional mutagenesis, some native strong promoters for foreign gene expression were found and examined. Several mutant strains expressing DB3 V_HR100/K were established, with the DB3 V_HR100/K coding region under the control of different promoters and at different insertion sites. In order to optimize the DB3 V_HR100/K production in cyanobacteria, different physiological parameters were tested including temperatures, light conditions, and different growth stages, also various purification methods of the expressed protein were investigated.

The ultimate goal of this study was to understand the antigen and antibody interaction of DB3 V_HR100/K produced in cyanobacteria. I demonstrated that the affinity and specificity of DB3 V_HR100/K generated in Synechocystis was closely similar profile to that produced in E. coli. However, in Synechocystis, the affinity of DB3 V_HR100/K with a His tag was much lower than that of DB3 V_HR100/K.