title: Production and purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro) with high-purity and low endotoxin content.
abstract: Streptococcus pneumoniae is the main cause of pneumonia, meningitis, and other conditions that kill thousands of children every year worldwide. The replacement of pneumococcal serotypes among the vaccinated population has evidenced the need for new vaccines with broader coverage and driven the research for protein-based vaccines. Pneumococcal surface protein A (PspA) protects S. pneumoniae from the bactericidal effect of human apolactoferrin and prevents complement deposition. Several studies indicate that PspA is a very promising target for novel vaccine formulations. Here we describe a production and purification process for an untagged recombinant fragment of PspA from clade 4 (PspA4Pro), which has been shown to be cross-reactive with several PspA variants. PspA4Pro was obtained using lactose as inducer in Phytone auto-induction batch or glycerol limited fed-batch in 5-L bioreactor. The purification process includes two novel steps: (i) clarification using a cationic detergent to precipitate contaminant proteins, nucleic acids, and other negatively charged molecules as the lipopolysaccharide, which is the major endotoxin; and (ii) cryoprecipitation that eliminates aggregates and contaminants, which precipitate at -20 °C and pH 4.0, leaving PspA4Pro in the supernatant. The final process consisted of cell rupture in a continuous high-pressure homogenizer, clarification, anion exchange chromatography, cryoprecipitation, and cation exchange chromatography. This process avoided costly tag removal steps and recovered 35.3 ± 2.5% of PspA4Pro with 97.8 ± 0.36% purity and reduced endotoxin concentration by >99.9%. Circular dichroism and lactoferrin binding assay showed that PspA4Pro secondary structure and biological activity were preserved after purification and remained stable in a wide range of temperatures and pH values.
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, Av Vital Brasil 1500, São Paulo, SP, 05503-900, Brazil.|Programa de Pós-Graduação Interunidades em Biotecnologia, Universidade de São Paulo, Avenida Prof. Lineu Prestes 2415, Edifício ICB-III, São Paulo, SP, 05508-900, Brazil.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Figueiredo
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, Av Vital Brasil 1500, São Paulo, SP, 05503-900, Brazil.
authors.forename: Eneas
authors.initials: E
authors.lastname: Carvalho
authors.affiliation: Departamento de Engenharia Química, Universidade Federal de São Carlos, Rodovia Washington Luís km 235, São Carlos, SP, 13565-905, Brazil.
authors.forename: Mauricio P
authors.initials: MP
authors.lastname: Santos
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, Av Vital Brasil 1500, São Paulo, SP, 05503-900, Brazil.|Programa de Pós-Graduação Interunidades em Biotecnologia, Universidade de São Paulo, Avenida Prof. Lineu Prestes 2415, Edifício ICB-III, São Paulo, SP, 05508-900, Brazil.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Kraschowetz
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, Av Vital Brasil 1500, São Paulo, SP, 05503-900, Brazil.|Programa de Pós-Graduação Interunidades em Biotecnologia, Universidade de São Paulo, Avenida Prof. Lineu Prestes 2415, Edifício ICB-III, São Paulo, SP, 05508-900, Brazil.
authors.forename: Rafaela T
authors.initials: RT
authors.lastname: Zanardo
authors.affiliation: Departamento de Engenharia Química, Universidade Federal de São Carlos, Rodovia Washington Luís km 235, São Carlos, SP, 13565-905, Brazil.
authors.forename: Gilson
authors.initials: G
authors.lastname: Campani
authors.affiliation: Departamento de Engenharia Química, Universidade Federal de São Carlos, Rodovia Washington Luís km 235, São Carlos, SP, 13565-905, Brazil.
authors.forename: Gabriel G
authors.initials: GG
authors.lastname: Silva
authors.affiliation: Departamento de Engenharia Química, Universidade Federal de São Carlos, Rodovia Washington Luís km 235, São Carlos, SP, 13565-905, Brazil.
authors.forename: Cíntia R
authors.initials: CR
authors.lastname: Sargo
authors.affiliation: Departamento de Engenharia Química, Universidade Federal de São Carlos, Rodovia Washington Luís km 235, São Carlos, SP, 13565-905, Brazil.
authors.forename: Antonio Carlos L
authors.initials: ACL
authors.lastname: Horta
authors.affiliation: Departamento de Engenharia Química, Universidade Federal de São Carlos, Rodovia Washington Luís km 235, São Carlos, SP, 13565-905, Brazil.
authors.forename: Roberto
authors.initials: R
authors.lastname: de C Giordano
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, Av Vital Brasil 1500, São Paulo, SP, 05503-900, Brazil.
authors.forename: Eliane N
authors.initials: EN
authors.lastname: Miyaji
authors.affiliation: Departamento de Engenharia Química, Universidade Federal de São Carlos, Rodovia Washington Luís km 235, São Carlos, SP, 13565-905, Brazil.
authors.forename: Teresa C
authors.initials: TC
authors.lastname: Zangirolami
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, Av Vital Brasil 1500, São Paulo, SP, 05503-900, Brazil.
authors.forename: Joaquin
authors.initials: J
authors.lastname: Cabrera-Crespo
authors.affiliation: Centro de Biotecnologia, Instituto Butantan, Av Vital Brasil 1500, São Paulo, SP, 05503-900, Brazil. viviane.goncalves@butantan.gov.br.
authors.forename: Viviane Maimoni
authors.initials: VM
authors.lastname: Gonçalves
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D003001:Cloning, Molecular; D003902:Detergents / Q000737:chemistry; D004731:Endotoxins / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D005990:Glycerol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007781:Lactoferrin / Q000737:chemistry; D007785:Lactose / Q000378:metabolism; D059625:Liquid-Liquid Extraction / Q000379:methods*; D011312:Pressure; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013296:Streptococcus pneumoniae / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D004731:Endotoxins; D011994:Recombinant Proteins; C051592:pneumococcal surface protein A; D007781:Lactoferrin; D007785:Lactose; D005990:Glycerol
keywords: Cetyltrimethylammonium bromide; Chromatography; Cryoprecipitation; Lactoferrin binding assay; Lipopolysaccharide removal; Streptococcus pneumoniae