Abstract
Streptococcus pneumoniae is a bacterium commonly found on human superior respiratory tract, colonizing nasopharynx, and is an important cause of pneumonia, otitis, meningitis and sepsis. All vaccines currently in use to fight pneumococcal diseases are based on the capsular polysaccharide (CP), plain or bound to a carrier protein. Unfortunately, due to their high prices, these vaccines are mostly unavailable to the majority of world population. Furthermore, vaccines available in Brazil are far from being able to cover all cases of pneumococcal diseases in the country. The use of pneumococcal proteins as carriers in a novel pneumococcal conjugate vaccine is an alternative to reduce the production costs, by including only CP from the most important serotypes in Brazil, and increase coverage due to the protection given by the protein moiety. Among candidate carrier proteins, pneumococcal surface protein A (PspA mas é da sigla em ingles!!!) has been shown promising results in animal essays of protection against challenge, and is highly conserved among pneumococcal strains: it can be classified in 3 families according to the amino acids sequence, and antibodies against PspA from one family cross reacts against other PspA from the same family. Moreover, PspA from families 1 and 2 were found in 95-99% of pneumococcal strains isolated from Brazilian population.One of the most important steps in vaccine production process is the purification of vaccine components, which must present a high degree of purity to satisfy the standards of regulatory agencies. Purification also represents an important part of the production costs. It involves a sequence of independent steps, which are based on the differences between physical-chemical properties of the product and the impurities, such as location in the cell, hydrophobicity, molecular weight, electric charge, etc., each step should progressively enrich the product end eliminate undesired components. Thus, this work aims to develop a purification process for a recombinant fragment of PspA from family 2, clade 4, containing the proline rich region (PspA4pro). PspA4pro has shown cross reactivity with family 2 as well as with some strains from family 1, it was obtained in high density cultures of E. coli and no fusions or tags were added to help its purification, which means that the development of this specific purification method will be a milestone on the development of processes to obtain recombinant bioproducts.The cultures in bioreactor for PspA4 production were performed by LaDaBio team, UFSCar, under supervision of Prof. Dr Teresa Cristina Zangirolami, and the cells were stored at -80ºC. The cell disruption will be carried out in a continuous high pressure homogenizer (APV Gaulin) with temperature control (~12ºC). The clarification will be done by centrifugation and clarified material will undergo the chromatographic steps. Ion exchange chromatography, hydrophobic interaction and gel filtration are going to be tested and different sequences are going to be evaluated in order to maximize yield and purity of the process. Purified protein will be characterized: PspA4pro molecular weight will be determined by HPSEC, the secondary structure will be analyzed by circular dichroism and Western Blotting will be used to verify the cross-recognition by antibodies against the native protein. (AU)
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