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.
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 K(2)HPO(4)/2.2 g/L KH(2)PO(4)), 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.
authors.affiliation: VDTEC-Vice-Diretoria de Desenvolvimento Tecnológico, Bio-Manguinhos/Fundação Oswaldo Cruz, Fiocruz, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ 21040-360, Brazil. ariane@bio.fiocruz.br
authors.forename: Ariane Leites
authors.initials: AL
authors.lastname: Larentis
authors.forename: Júlia Fabiana Monteiro Quintal
authors.initials: JF
authors.lastname: Nicolau
authors.forename: Ana Paula Corrêa
authors.initials: AP
authors.lastname: Argondizzo
authors.forename: Ricardo
authors.initials: R
authors.lastname: Galler
authors.forename: Maria Isabel
authors.initials: MI
authors.lastname: Rodrigues
authors.forename: Marco Alberto
authors.initials: MA
authors.lastname: Medeiros
mesh_terms: D018829:Adhesins, Bacterial / Q000096:biosynthesis*; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005990:Glycerol / Q000378:metabolism; D008074:Lipoproteins / Q000096:biosynthesis*; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000235:genetics
chemical_list: D018829:Adhesins, Bacterial; D003470:Culture Media; D008074:Lipoproteins; C085000:PsaA protein, Streptococcus; D005990:Glycerol