Association of high pressure and alkaline condition for solubilization of inclusion bodies and refolding of the NS1 protein from zika virus

Autor(es)

Rosa da Silva, Cleide Mara ^[1] ; Chura-Chambi, Rosa Maria ^[1] ; Pereira, Lennon Ramos ^[2] ; Cordeiro, Yraima ^[3] ; de Souza Ferreira, Luis Carlos ^[2] ; Morganti, Ligia ^[1]

Afiliação do(s) autor(es)

^[1] IPEN, CNEN, Ctr Biotecnol, SP, Av Prof Lineu Prestes 2242, BR-05508000 Sao Paulo - Brazil

^[2] Univ Sao Paulo, Inst Ciencias Biomed, Dept Microbiol, Av Prof Lineu Prestes 1374, BR-05508000 Sao Paulo - Brazil

^[3] Univ Fed Rio de Janeiro, Fac Farm, Av Carlos Chagas Filho 373, BR-21941902 Rio De Janeiro - Brazil

• Tipo de documento: Artigo Científico
• Fonte: BMC Biotechnology; v. 18, DEC 12 2018.
• Citações Web of Science: 1

Resumo

BackgroundProteins 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.ResultsPressure-treatment (2.4kbar) 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 pH8.5, NS1 was refolded and formed soluble oligomers. High (up to 68mg/liter) NS1 concentrations were obtained by solubilization of NS1-IBs at pH11 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.ConclusionsThe 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. (AU)

• Processo FAPESP: 15/02574-0 - Renaturação de antígenos sintetizados como corpos de inclusão em Escherichia coli para preparação de vacinas
• Beneficiário: Ligia Ely Morganti Ferreira Dias
• Modalidade de apoio: Auxílio à Pesquisa - Regular