Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/32345
Title: Optimization of medium formulation and seed conditions for expression of mature PsaA (pneumococcal surface adhesin A) in Escherichia coli using a sequential experimental design strategy and response surface methodology
Authors: Larentis, Ariane Leites
Nicolau, Júlia Fabiana Monteiro Quintal
Argondizzo, Ana Paula Corrêa
Galler, Ricardo
Rodrigues, Maria Isabel
Medeiros, Marco Alberto
Affilliation: Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.
Universidade Estadual de Campinas. Faculdade de Engenharia de Alimentos. Campinas, Brasil / PROTIMIZA Consulting and Training in Experimental Design and Process Optimization. Campinas, Brasil.
Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.
Abstract: PsaA, a candidate antigen for a vaccine against pneumonia, is well-conserved in all Streptococcus pneumoniae serotypes. A sequence of two-level experimental designs was used to evaluate medium composition and seed conditions to optimize the expression of soluble mature PsaA in E. coli. A face-centered central composite design was first used to evaluate the effects of yeast extract (5 and 23.6 g/L), tryptone (0 and 10 g/L), and glucose (1 and 10 g/L), with replicate experiments at the central point (14.3 g/L yeast extract, 5 g/L tryptone, 5.5 g/L glucose). Next, a central composite design was used to analyze the influence of NaCl concentration (0, 5, and 10 g/L) compared with potassium salts (9.4 g/L K2HPO4/2.2 g/L KH2PO4), and seed growth (7 and 16 h). Tryptone had no significant effect and was removed from the medium. Yeast extract and glucose were optimized at their intermediate concentrations, resulting in an animal-derived material-free culture medium containing 15 g/L yeast extract, 8 g/L glucose, 50 μg/mL kanamycin, and 0.4% glycerol, yielding 1 g/L rPsaA after 16 h induction at 25°C in shake flasks at 200 rpm. All the seed age and salt conditions produced similar yields, indicating that no variation had a statistically significant effect on expression. Instead of growing the seed culture for 16 h (until saturation), the process can be conducted with 7 h seed growth until the exponential phase. These results enhanced the process productivity and reduced costs, with 5 g/L NaCl being used rather than potassium salts.
Keywords: Design of experiments (DoE)
Modeling
Optimization
Response surface methodology (RSM)
Statistical experimental design
Issue Date: 2012
Citation: LARENTIS, Ariane Leites et al. Optimization of medium formulation and seed conditions for expression of mature PsaA (pneumococcal surface adhesin A) in Escherichia coli using a sequential experimental design strategy and response surface methodology. Journal of Industrial Microbiology & Biotechnology, Houndmills, v. 39, n. 6, p. 897-908, 2012.
DOI: 10.1007/s10295-012-1099-8
ISSN: 1476-5535
Copyright: restricted access
Appears in Collections:Biomanguinhos - Artigos de Periódicos

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