Production and purification of recombinant fragment of pneumococcal surface protein A (PspA) in Escherichia coli

Barazzone, Giovana Cappio; Carvalho, Rimenys J.; Kraschowetz, Stefanie; Horta, Antonio Carlos Luperni; Sargo, Cíntia Regina; Silva, Adilson J.; Zangirolami, Teresa Cristina; Goulart, Cibelly; Leite, Luciana C. C.; Tanizaki, Martha M.; Gonçalves, Viviane Maimoni; Cabrera-Crespo, Joaquín

doi:10.1016/j.provac.2011.07.005

Cited by 11 publications

(4 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The NH 2 , a nucleophilic group from PS-OCT (C), reacts with the intermediate (A-B) through a carboxamide linkage, generating the conjugate (A-C). The use of this reagent in the production of a conjugate vaccine was first proposed by our laboratory (29). The majority of the conjugation methods that are based on the reaction of carboxyl groups employ 1-ethyl-3-(3-dimethylaminpropyl) carbodiimide hydrochloride (EDC) in their activation step.…”

Section: Discussionmentioning

confidence: 99%

Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response

Perciani

Barazzone

Goulart

et al. 2013

Clin Vaccine Immunol

Self Cite

View full text Add to dashboard Cite

bDespite the substantial beneficial effects of incorporating the 7-valent pneumococcal conjugate vaccine (PCV7) into immunization programs, serotype replacement has been observed after its widespread use. As there are many serotypes currently documented, the use of a conjugate vaccine relying on protective pneumococcal proteins as active carriers is a promising alternative to expand PCV coverage. In this study, capsular polysaccharide serotype 6B (PS6B) and recombinant pneumococcal surface protein A (rPspA), a well-known protective antigen from Streptococcus pneumoniae, were covalently attached by two conjugation methods. The conjugation methodology developed by our laboratory, employing 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as an activating agent through carboxamide formation, was compared with reductive amination, a classical methodology. DMT-MM-mediated conjugation was shown to be more efficient in coupling PS6B to rPspA clade 1 (rPspA1): 55.0% of PS6B was in the conjugate fraction, whereas 24% was observed in the conjugate fraction with reductive amination. The influence of the conjugation process on the rPspA1 structure was assessed by circular dichroism. According to our results, both conjugation processes reduced the alpha-helical content of rPspA; reduction was more pronounced when the reaction between the polysaccharide capsule and rPspA1 was promoted between the carboxyl groups than the amine groups (46% and 13%, respectively). Regarding the immune response, both conjugates induced functional anti-rPspA1 and anti-PS6B antibodies. These results suggest that the secondary structure of PspA1, as well as its reactive groups (amine or carboxyl) involved in the linkage to PS6B, may not play an important role in eliciting a protective immune response to the antigens.

show abstract

Section: Discussionmentioning

confidence: 99%

Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response

Perciani

Barazzone

Goulart

et al. 2013

Clin Vaccine Immunol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Two new constructs were proposed, which would join the antigens with different molecular linkers: A flexible linker (FL), formed by glycines and serines, whose sequence is N- GGGGSGGGGS- C; and a rigid linker (RL), which forms an alpha-helix structure, whose sequence is N- AEAAAKEAAAKA -C. In addition, within the proline-rich region, the non-proline sequence block (NonPro), and the last block of prolines of PspA were removed from this new construct ( Hollingshead et al, 2000 ), since recombinant PspA molecules that do not have this non-Pro region, such as PspA4Pro ( Moreno et al, 2010 ; Figueiredo et al, 2017 ) and PspA3 ( Carvalho et al, 2012 ), were more stable than recombinant PspA245 in whose sequence the region was maintained ( Barazzone et al, 2011 ). Also, the new constructs were cloned into pET28a, eliminating nine pQE-30 derived amino acids from beginning of the tandem fusion protein, three (RGS) before six histidine tag (His-tag), and another six (GSACEL) after His-tag and before the first PspA amino acid.…”

Section: Methodsmentioning

confidence: 99%

Peptide linker increased the stability of pneumococcal fusion protein vaccine candidate

Zane

Kraschowetz

Trentini

et al. 2023

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

Streptococcus pneumoniae is a bacterial pathogen exclusive to humans, responsible for respiratory and systemic diseases. Pneumococcal protein vaccines have been proposed as serotype-independent alternatives to currently used conjugated polysaccharide vaccines, which have presented limitations regarding their coverage. Previously in our group, pneumococcal surface protein A (PspA) and detoxified pneumolysin (PdT) were genetically fused and the hybrid protein protected mice against pneumococcal challenge, offered higher cross-protection against different strains and showed greater opsonophagocytosis rate than co-administered proteins. As juxtaposed fusion was unstable to upscale production of the protein, flexible (PspA-FL-PdT) and rigid (PspA-RL-PdT) molecular linkers were inserted between the antigens to increase stability. This work aimed to produce recombinant fusion proteins, evaluate their stability after linker insertion, both in silico and experimentally, and enable the production of two antigens in a single process. The two constructs with linkers were cloned into Escherichia coli and hybrid proteins were purified using chromatography; purity was evaluated by SDS-PAGE and stability by Western blot and high performance size exclusion chromatography. PspA-FL-PdT showed higher stability at −20°C and 4°C, without additional preservatives. In silico analyses also showed differences regarding stability of the fusion proteins, with molecule without linker presenting disallowed amino acid positions in Ramachandran plot and PspA-FL-PdT showing the best scores, in agreement with experimental results. Mice were immunized with three doses and different amounts of each protein. Both fusion proteins protected all groups of mice against intranasal lethal challenge. The results show the importance of hybrid protein structure on the stability of the products, which is essential for a successful bioprocess development.

show abstract

“…Ongoing cell disruption is performed with high-pressure homogenization, as high-pressure homogenization is the only scalable form of cell disruption (Balasundaram et al, 2009; Lin and Cai, 2009). Homogenizers can be operated at high velocities; realizing cell disruption within one passage and the implementation of a continuous cell disruption mode is rather easy (Barazzone et al, 2011). The following centrifugation step, separating cell debris from soluble proteins, is also feasible in a continuous mode with techniques available nowadays (Palmer and Wingfield, 2012; Chatel et al, 2014).…”

Section: The Continuous Downstream Processing Of Intracellular Proteinsmentioning

confidence: 99%

The Rocky Road From Fed-Batch to Continuous Processing With E. coli

Kopp

Slouka²,

Spadiut³

et al. 2019

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

Escherichia coli still serves as a beloved workhorse for the production of many biopharmaceuticals as it fulfills essential criteria, such as having fast doubling times, exhibiting a low risk of contamination, and being easy to upscale. Most industrial processes in E. coli are carried out in fed-batch mode. However, recent trends show that the biotech industry is moving toward time-independent processing, trying to improve the space–time yield, and especially targeting constant quality attributes. In the 1950s, the term “chemostat” was introduced for the first time by Novick and Szilard, who followed up on the previous work performed by Monod. Chemostat processing resulted in a major hype 10 years after its official introduction. However, enthusiasm decreased as experiments suffered from genetic instabilities and physiology issues. Major improvements in strain engineering and the usage of tunable promotor systems facilitated chemostat processes. In addition, critical process parameters have been identified, and the effects they have on diverse quality attributes are understood in much more depth, thereby easing process control. By pooling the knowledge gained throughout the recent years, new applications, such as parallelization, cascade processing, and population controls, are applied nowadays. However, to control the highly heterogeneous cultivation broth to achieve stable productivity throughout long-term cultivations is still tricky. Within this review, we discuss the current state of E. coli fed-batch process understanding and its tech transfer potential within continuous processing. Furthermore, the achievements in the continuous upstream applications of E. coli and the continuous downstream processing of intracellular proteins will be discussed.

show abstract

Production and purification of recombinant fragment of pneumococcal surface protein A (PspA) in Escherichia coli

Cited by 11 publications

References 18 publications

Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response

Conjugation of Polysaccharide 6B from Streptococcus pneumoniae with Pneumococcal Surface Protein A: PspA Conformation and Its Effect on the Immune Response

Peptide linker increased the stability of pneumococcal fusion protein vaccine candidate

The Rocky Road From Fed-Batch to Continuous Processing With E. coli

Contact Info

Product

Resources

About