title: Experimental design approach in recombinant protein expression: determining medium composition and induction conditions for expression of pneumolysin from Streptococcus pneumoniae in Escherichia coli and preliminary purification process.
abstract: BACKGROUND
  Streptococcus pneumoniae (S. pneumoniae) causes several serious diseases including pneumonia, septicemia and meningitis. The World Health Organization estimates that streptococcal pneumonia is the cause of approximately 1.9 million deaths of children under five years of age each year. The large number of serotypes underlying the disease spectrum, which would be reflected in the high production cost of a commercial vaccine effective to protect against all of them and the higher level of amino acid sequence conservation as compared to polysaccharide structure, has prompted us to attempt to use conserved proteins for the development of a simpler vaccine. One of the most prominent proteins is pneumolysin (Ply), present in almost all the serotypes known at the moment, which shows an effective protection against S. pneumoniae infections.
  
  
  RESULTS
  We have cloned the pneumolysin gene from S. pneumoniae serotype 14 and studied the effects of eight variables related to medium composition and induction conditions on the soluble expression of rPly in Escherichia coli (E. coli) and a 28-4 factorial design was applied. Statistical analysis was carried out to compare the conditions used to evaluate the expression of soluble pneumolysin; rPly activity was evaluated by hemolytic activity assay and served as the main response to evaluate the proper protein expression and folding. The optimized conditions, validated by the use of triplicates, include growth until an absorbance of 0.8 (measured at 600 nm) with 0.1 mM IPTG during 4 h at 25°C in a 5 g/L yeast extract, 5 g/L tryptone, 10 g/L NaCl, 1 g/L glucose medium, with addition of 30 μg/mL kanamycin.
  
  
  CONCLUSIONS
  This experimental design methodology allowed the development of an adequate process condition to attain high levels (250 mg/L) of soluble expression of functional rPly in E. coli, which should contribute to reduce operational costs. It was possible to recover the protein in its active form with 75% homogeneity.
authors.affiliation: Bio-Manguinhos (Instituto de Tecnologia em Imunobiológicos) - Fundação Oswaldo Cruz (FIOCRUZ) - VDTEC (Vice-Diretoria de Desenvolvimento Tecnológico), Av, Brasil 4365, Pavilhão Rockfeller Sala 202 - 21040-360, Manguinhos, Rio de Janeiro, RJ, Brazil. guillermo@peq.coppe.ufrj.br.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Marini
authors.forename: Mateus Dalcin
authors.initials: MD
authors.lastname: Luchese
authors.forename: Ana Paula Correa
authors.initials: AP
authors.lastname: Argondizzo
authors.forename: Ana Carolina Magalhães Andrade
authors.initials: AC
authors.lastname: de Góes
authors.forename: Ricardo
authors.initials: R
authors.lastname: Galler
authors.forename: Tito Lívio Moitinho
authors.initials: TL
authors.lastname: Alves
authors.forename: Marco Alberto
authors.initials: MA
authors.lastname: Medeiros
authors.forename: Ariane Leites
authors.initials: AL
authors.lastname: Larentis
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D003627:Data Interpretation, Statistical; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015999:Multivariate Analysis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015203:Reproducibility of Results; D012107:Research Design; D013296:Streptococcus pneumoniae; D013301:Streptolysins / Q000096:biosynthesis*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae