title: Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage.
abstract: The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.
authors.affiliation: Fundação Oswaldo Cruz (Fiocruz), Bio-Manguinhos/VDTEC (Vice-Diretoria de Desenvolvimento Tecnológico), Laboratório de Tecnologia Recombinante (LATER), Av. Brasil 4365, 21.040-360, Rio de Janeiro - RJ, Brazil. ariane@bio.fiocruz.br
authors.forename: Ariane Leites
authors.initials: AL
authors.lastname: Larentis
authors.forename: Ana Paula Corrêa
authors.initials: AP
authors.lastname: Argondizzo
authors.forename: Gabriela dos Santos
authors.initials: Gdos S
authors.lastname: Esteves
authors.forename: Ellen
authors.initials: E
authors.lastname: Jessouron
authors.forename: Ricardo
authors.initials: R
authors.lastname: Galler
authors.forename: Marco Alberto
authors.initials: MA
authors.lastname: Medeiros
mesh_terms: D018829:Adhesins, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods*; D004356:Drug Storage; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005947:Glucose; D007544:Isopropyl Thiogalactoside; D008074:Lipoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D013696:Temperature
chemical_list: D018829:Adhesins, Bacterial; D008074:Lipoproteins; C085000:PsaA protein, Streptococcus; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D005947:Glucose