title: Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences.
abstract: Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.
authors.affiliation: Laboratório de Bioprocessos, Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil.
authors.forename: Rimenys Junior
authors.initials: RJ
authors.lastname: Carvalho
authors.forename: Joaquin
authors.initials: J
authors.lastname: Cabrera-Crespo
authors.forename: Martha Massako
authors.initials: MM
authors.lastname: Tanizaki
authors.forename: Viviane Maimoni
authors.initials: VM
authors.lastname: Gonçalves
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D002244:Carbon / Q000378:metabolism*; D002845:Chromatography / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; C051592:pneumococcal surface protein A; D002244:Carbon