even with 40% segregation, phytase production continued to rise. After two and a half hours’ induction, phytase production rose again to 1000 U/L, while segregation increased to 80%. It was only after this point that phytase activity started to drop [33]. The data presented in Fig. 5 show that after 4 h induction the fraction of plasmid-bearing cells stood at around 45%,

while the yield factor was still rising. However, as shown by other authors [33], if segregation were to rise even higher, the yield factor could start to fall. High levels of a soluble form of ClpP were expressed in all the experiments from the experimental design used. Plasmid segregation was identified in the system throughout the kanamycin concentration range tested. The lowest concentration of IPTG (0.1 mM) tested in this Forskolin study resulted in greater plasmid ABT263 stability. The statistical analyses made of the procedures used to determine plasmid segregation confirmed that they are reproducible. By using experimental design it was possible to conclude that the optimal point of the system was with 0.1 mM IPTG and 0 μg/mL kanamycin, which yielded 247.3 mg/L ClpP; this optimal condition was validated with success. It should therefore be possible to reduce the inducer concentration tenfold and eliminate the antibiotic from the system while still keeping

protein expression at similar levels and reducing overall process costs. It is also important to highlight the importance of the study of plasmid segregation in recombinant systems, since plasmid stability is one of the lynchpins of recombinant protein production. Experimental design proved to be a powerful tool for determining the optimal conditions for expressing recombinant Dichloromethane dehalogenase protein in E. coli using a minimum number of experiments, enabling an assessment to be made of the effect of each of the

variables, their interactions and experimental errors. It is still common practice in molecular biology for each variable to be evaluated separately, which may result in misinterpretations of the data obtained, because it fails to take account of their interactions. Experimental design enables the selection of the best test conditions for detecting the interactions between the variables, which is not possible empirically by adopting the methods usually used in the area that treat variables independently. These techniques have universal application in the production of recombinant proteins. This work received financial support from Bio-Manguinhos and PAPES V (Programa Estratégico de Apoio à Pesquisa em Saúde) from Fundação Oswaldo Cruz (FIOCRUZ). Karen Einsfeldt and João B. Severo Júnior received scholarships from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), respectively.