ROSA MARIA CHURA CHAMBI

ROSA MARIA CHURA CHAMBI

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Protein refolding based on high hydrostatic pressure and alkaline pH
2019 - CHURA-CHAMBI, ROSA M.; SILVA, CLEIDE M.R. da; PEREIRA, LENNON R.; BARTOLINI, PAOLO; FERREIRA, LUIS C. de S.; MORGANTI, LIGIA
In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion bodies (IBs) generated by recombinant proteins expressed by Escherichia coli strains. The method was successfully applied to a recombinant form of the dengue virus (DENV) non-structural protein 1 (NS1). The minimal pH for IBs solubilization at 1 bar was 12 while a pH of 10 was sufficient for solubilization at HHP: 2.4 kbar for 90 min and 0.4 kbar for 14 h 30 min. An optimal refolding condition was achieved by compression of IBs at HHP and pH 10.5 in the presence of arginine, oxidized and reduced glutathiones, providing much higher yields (up to 8-fold) than association of HHP and GdnHCl via an established protocol. The refolded NS1, 109 ± 9.5 mg/L bacterial culture was recovered mainly as monomer and dimer, corresponding up to 90% of the total protein and remaining immunologically active. The proposed conditions represent an alternative for the refolding of immunologically active recombinant proteins expressed as IBs.
Association of high pressure and alkaline condition for solubilization of inclusion bodies and refolding of the NS1 protein from zika virus
2018 - SILVA, CLEIDE M.R. da; CHURA-CHAMBI, ROSA M.; PEREIRA, LENNON R.; CORDEIRO, YRAIMA; FERREIRA, LUIS C. de S.; MORGANTI, LIGIA
Background: Proteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures. Removal of the chaotropes frequently causes reaggregation and poor recovery of bioactive proteins. High hydrostatic pressure (HHP) and alkaline pH are two conditions that, in the presence of low level of chaotropes, have been described as non-denaturing solubilization agents. In the present study we evaluated the strategy of combination of HHP and alkaline pH on the solubilization of IB using as a model an antigenic form of the zika virus (ZIKV) non-structural 1 (NS1) protein. Results: Pressure-treatment (2.4 kbar) of NS1-IBs at a pH of 11.0 induced a low degree of NS1 unfolding and led to solubilization of the IBs, mainly into monomers. After dialysis at pH 8.5, NS1 was refolded and formed soluble oligomers. High (up to 68 mg/liter) NS1 concentrations were obtained by solubilization of NS1-IBs at pH 11 in the presence of arginine (Arg) with a final yield of approximately 80% of total protein content. The process proved to be efficient, quick and did not require further purification steps. Refolded NS1 preserved biological features regarding reactivity with antigen-specific antibodies, including sera of ZIKV-infected patients. The method resulted in an increase of approximately 30-fold over conventional IB solubilization-refolding methods. Conclusions: The present results represent an innovative non-denaturing protein refolding process by means of the concomitant use of HHP and alkaline pH. Application of the reported method allowed the recovery of ZIKV NS1 at a condition that maintained the antigenic properties of the protein.
Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3
2018 - CHURA-CHAMBI, ROSA M.; FRAGA, TATIANA R.; SILVA, LUDMILA B. da; YAMAMOTO, BRUNO B.; ISAAC, LOURDES; BARBOSA, ANGELA S.; MORGANTI, LIGIA
Enzymes from the thermolysin family are crucial factors in the pathogenesis of several diseases caused by bacteria and are potential targets for therapeutic interventions. Thermolysin encoded by the gene LIC13322 of the causative agent of leptospirosis, Leptospira interrogans, was shown to cleave proteins from the Complement System. However, the production of this recombinant protein using traditional refolding processes with high levels of denaturing reagents for thermolysin inclusion bodies (TL-IBs) solubilization results in poor recovery and low proteolytic activity probably due to improper refolding of the protein. Based on the assumption that leptospiral proteases play a crucial role during infection, the aim of this work was to obtain a functional recombinant thermolysin for future studies on the role of these metalloproteases on leptospiral infection. The association of high hydrostatic pressure (HHP) and alkaline pH was utilized for thermolysin refolding. Incubation of a suspension of TL-IBs at HHP and a pH of 11.0 is non-denaturing but effective for thermolysin solubilization. Soluble protein does not reaggregate by dialysis to pH 8.0. A volumetric yield of 46 mg thermolysin/L of bacterial culture and a yield of near 100% in relation to the total thermolysin present in TL-IBs were obtained. SEC-purified thermolysin suffers fragmentation, likely due to autoproteolysis and presents proteolytic activity against complement C3 α-chain, possibly by a generation of a C3b-like molecule. The proteolytic activity of thermolysin against C3 was time and dose-dependent. The experience gained in this study shall help to establish efficient HHP-based processes for refolding of bioactive proteins from IBs.
Investigation on solubilization protocols in the refolding of the thiredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach
2015 - LEMKE, LAURA S.; CHURA-CHAMBI, ROSA M.; RODRIGUES, DANIELLA; CUSSIOL, JOSE R.R.; MALAVASI, NATALIA V.; ALEGRIA, THIAGO G.P.; SOARES NETTO, LUIS E.; MORGANTI, LIGIA
High-pressure refolding of aggregated fusion proteins: endostatin-proapoptotic peptides with enhanced antiangiogenic activity
2008 - CHURA-CHAMBI, R.M.; AFFONSO, R.; GENOVA, L.; MORGANTI, L.
Application of high hydrostatic pressure for recovery of biologically active cruzain from recombinant inclusion bodies
2011 - CHAMBI, ROSA M.C.; LEMKE, LAURA S.; VALLEJO, NATALIA M.; JULIANO NETO, LUIZ; MORGANTI, LIGIA
Refolding of the recombinant protein Sm29, a step toward the production of the vaccine candidate against schistosomiases
2013 - CHURA-CHAMBI, ROSA M.; NAKAJIMA, ERIKA; CARVALHO, ROBERTA R. de; MIYASATO, PATRICIA A.; OLIVEIRA, SÉRGIO C.; MORGANTI, LIGIA; MARTINS, ELIZABETH A.L.
An analysis of the factors that affect the dissociation of inclusion bodies and the refolding of endostatin under high pressure
2013 - CHURA-CHAMBI, R.M.; CORDEIRO, Y.; MALAVASI, N.V.; LEMKE, L.S.; RODRIGUES, D.; MORGANTI, L.
Refolding of endostatin from inclusion bodies using high hydrostatic pressure
2008 - CHURA-CHAMBI, ROSA M.; GENOVA, LUIS A.; AFFONSO, REGINA; MORGANTI, LIGIA
Refolding on the recombinant protein OmpA70 from Leptospira interrogans from inclusion bodies using high hydrostatic pressure and partial characterization of its immunological properties
2010 - FRAGA, TATIANA R.; CHURA-CHAMBI, ROSA M.; GONCALEZ, AMANE P.; MORAIS, ZENAIDE M.; VASCONCELLOS, SILVIO A.; MORGANTI, LIGIA; MARTINS, ELIZABETH A.L.