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Production and purification of recombinant EDIII ZIKV using CASPON technology in E. coli expression system

Grant number: 21/08502-1
Support type:Scholarships abroad - Research Internship - Doctorate (Direct)
Effective date (Start): October 18, 2021
Effective date (End): October 17, 2022
Field of knowledge:Engineering - Chemical Engineering
Principal researcher:Viviane Maimoni Gonçalves
Grantee:Sérgio Carneiro Araújo
Supervisor abroad: Alois Jungbauer
Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
Research place: Universität für Bodenkultur Wien, Austria  
Associated to the scholarship:18/24683-3 - Production of envelope protein domain III of Zika Virus in Escherichia coli: evaluation of the protein obtained from soluble fraction and inclusion bodies, BP.DD

Abstract

EDIII is the most exposed region of Zika Virus E protein and evokes potentneutralizing antibodies. It has the required characteristics to be used as a safe andeffective antigen for a subunit Zika vaccine. EDIII production in E. coli has beenreported only at lab-scale and mostly refolded from inclusion bodies (IB), which isdifficult to scale-up maintaining protein function. Fusion tag technology has beenused to avoid IB formation and to favor protein stability and proper biological activity.This technology was used by the PhD. student for EDIII production. Although theexpressive solubility improvement, instability and degradation have been observedafter EDIII purification and tag removal. This may be caused by the non-native aminoacid that the TEV protease leaves at the N-terminus of EDIII after tag removal. TheInstitute of Bioprocess Science and Engineering located in Austria has been workingfor decades creating a fusion tag platform that fulfils the requirements for industrialapplications. They recently developed CASPON, an affinity Fusion-Tag platform witha completely removable N-terminal tag. It consists of the T7AC solubility tag, a6-histidine tag, a short linker and the caspase-2 cleavage site. Initial studies usingCASPON have demonstrated considerable solubility and yield increase using thistechnology for production of proteins with intrinsic tendency to form IB. In this BEPEproject, we propose to study the recently developed CASPON technology for EDIIIproduction in E. coli. This study can increase EDIII production yield and stability, aidCASPON validation and comparison with the Fh8-TEV platform used here. Besides,the high quality and expertise of the host group will contribute to deepening thestudent PhD. formation and to advance his research project.

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