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
1 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
2 Programa de Engenharia Química - COPPE - Universidade Federal do Rio de Janeiro (UFRJ), Av. Horácio Macedo 2030, Bloco G, Sala 115 - 21941-972, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
3 ENSP (Escola Nacional de Saúde Pública Sergio Arouca) - Fundação Oswaldo Cruz (FIOCRUZ) - Centro de Estudos da Saúde do Trabalhador e Ecologia Humana (CESTEH), Av. Leopoldo Bulhões 1480, Prédio 1º de Maio - 21041-210, Manguinhos, Rio de Janeiro, RJ, Brazil
BMC Biotechnology 2014, 14:1 doi:10.1186/1472-6750-14-1Published: 9 January 2014
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.
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.
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.