Experimental design approach in recombinant protein expression: determining medium composition and induction conditions for expression of pneumolysin from Streptococcus pneumoniae in Escherichia coliand preliminary purification process

Abstract

BackgroundStreptococcus 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.ResultsWe 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.ConclusionsThis 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.

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells

“…CHEF1-based expression vector containing the 4.1 kb upstream and 4.2 kb downstream flanking regions of the CHO-EF1 gene was used to express recombinant proteins in mammalian cells [9]. Although these flanking sequences improve expression considerably, their incorporation into the promoter region leads to an increase in the vector size which could cause difficulties in cells transfection and genomic stability [27]. Formerly, the efficiency of CHEF promoter region excluding flanking regions was examined for expression of enhanced green fluorescent protein, EGFP, in stem cells and CHO-K1 cells.…”

Recombinant H7 hemagglutinin forms subviral particles that protect mice and ferrets from challenge with H7N9 influenza virus

“…Studies have shown that immune response can depend on the molecular structure of recombinant HA protein [28]. A previous study has shown that purified rH3 HA forms rosette-shaped subviral particles (SVPs) of approximately 30–50 nm in diameter [29]. However, it is not clear if such SVPs are also formed by rH7 HA and if rH7 HA SVPs are capable of inducing protective immunity in animal models.…”

Recombinant Hemagglutinin and Virus-Like Particle Vaccines for H7N9 Influenza virus

“…However, rHA is also immunogenic and protective in animal models and in humans [61,64]. Immunogenicity of rHA at least in part can be explained by the observed arrangement of rHA into subviral particles, SVP, with up to 30–50 nm in diameter and containing multiple rHA molecules [66]. Thus, it appears that successful recombinant vaccines including L1-based HPV, HBsAg-based HBV, and even rHA-based influenza represent particulate structures such as VLPs or SVP, with oligomeric/particulate structures likely responsible for high immunogenicity of these vaccines.…”

“…A previous study has shown that purified rH3 HA forms rosette-shaped subviral particles (SVPs) of approximately 30–50 nm in diameter [66]. We recently prepared the full-length H7 rHA from A/Anhui/1/2013 (H7N9) using a modified purification conditions for rH7 HA [67].…”