Efficient recovery of recombinant CRM197 expressed as inclusion bodies in E.coli.
PLoS One
; 13(7): e0201060, 2018.
Article
em En
| MEDLINE
| ID: mdl-30021008
ABSTRACT
CRM197, which retains the same inflammatory and immune-stimulant properties as diphtheria toxin but with reduced toxicity, has been used as a safe carrier in conjugated vaccines. Expression of recombinant CRM197 in E. coli is limited due to formation of inclusion bodies. Soluble expression attempts in Bacillus subtilis, P. fluorescens, Pichia pastoris, and E. coli were partially unsuccessful or did not generate yields sufficient for industrial scale production. Multiple approaches have been attempted to produce CRM197 in E. coli, which has attractive features such as high yield, simplicity, fast growth, etc., including expression of oxidative host, concurrent expression of chaperones, or periplasmic export. Recently, alternative methods for recovery of insoluble proteins expressed in E. coli were reported. Compared to traditional denaturation/refolding, these methods used the non-denaturing solubilization agent, N-lauroylsarkosine to obtain higher recovery yields of native proteins. Based on this work, here, we focused on solubilization of CRM197 from E. coli inclusion bodies. First, CRM197 was expressed as inclusion bodies by high-level expression of recombinant CRM197 in E. coli (126.8 mg/g dcw). Then bioactive CRM197 was isolated from these inclusion bodies with high yield (108.1 mg/g dcw) through solubilization with N-lauroylsarkosine including Triton X-100 and CHAPS, and purified by Ni-affinity chromatography and size-exclusion chromatography. In this study, we present a cost-effective alternative for the production of bioactive CRM197 and compare our recovery yield with yields in other production processes.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
/
Proteínas Recombinantes
/
Corpos de Inclusão
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Escherichia coli
Idioma:
En
Revista:
PLoS One
Assunto da revista:
CIENCIA
/
MEDICINA
Ano de publicação:
2018
Tipo de documento:
Article