Virus-like particle preparation is improved by control over capsomere-DNA interactions during chromatographic purification.
Biotechnol Bioeng
; 118(4): 1707-1720, 2021 04.
Article
em En
| MEDLINE
| ID: mdl-33484156
ABSTRACT
Expression of viral capsomeres in bacterial systems and subsequent in vitro assembly into virus-like particles is a possible pathway for affordable future vaccines. However, purification is challenging as viral capsomeres show poor binding to chromatography media. In this study, the behavior of capsomeres in unfractionated bacterial lysate was compared with that for purified capsomeres, with or without added microbial DNA, to better understand reasons for poor bioprocess behavior. We show that aggregates or complexes form through the interaction between viral capsomeres and DNA, especially in bacterial lysates rich in contaminating DNA. The formation of these complexes prevents the target protein capsomeres from accessing the pores of chromatography media. We find that protein-DNA interactions can be modulated by controlling the ionic strength of the buffer and that at elevated ionic strengths the protein-DNA complexes dissociate. Capsomeres thus released show enhanced bind-elute behavior on salt-tolerant chromatography media. DNA could therefore be efficiently removed. We believe this is the first report of the use of an optimized salt concentration that dissociates capsomere-DNA complexes yet enables binding to salt-tolerant media. Post purification, assembly experiments indicate that DNA-protein interactions can play a negative role during in vitro assembly, as DNA-protein complexes could not be assembled into virus-like particles, but formed worm-like structures. This study reveals that the control over DNA-protein interaction is a critical consideration during downstream process development for viral vaccines.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Contexto em Saúde:
3_ND
Problema de saúde:
3_neglected_diseases
/
3_zoonosis
Assunto principal:
DNA Bacteriano
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Proteínas do Capsídeo
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Escherichia coli
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Vacinas de Partículas Semelhantes a Vírus
Idioma:
En
Revista:
Biotechnol Bioeng
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Austrália