Trial and error: New insights into recombinant expression of membrane-bound insect cytochromes P450 in Escherichia coli systems.
Int J Biol Macromol
; 273(Pt 2): 133183, 2024 Jul.
Article
em En
| MEDLINE
| ID: mdl-38897522
ABSTRACT
Insect cytochromes P450 (CYP450s) are key enzymes responsible for a wide array of oxidative transformations of both endogenous and exogenous substrates. However, there is currently no a universal guideline established for heterologous expression of membrane-bound CYP450s, which hampers their downstream biochemical and structural studies. In this study, we conducted large-scale screening of protein overexpression in Escherichia coli using 71 insect CYP450 sequences and optimized the expression of a difficult-to-express CYP450 (CYP6HX3) using eight different optimizations, including selection of host strains and expression vectors, alternative of leader signal peptides, and N-terminal modifications. We confirmed that 1) Only insect CYP450s belonging to the CYP347 family could be expressed with N-terminal fusion of ompA2+ signal peptide in E. coli expression system. 2) E. coli Lemo 21 (DE3) effectively improved the expression of CYP6HX3 in the plasma membrane. 3) A brick-red appearance occurred frequently in the expressed thallus or membrane proteins, but this phenomenon could not necessarily indicate successful overexpression of target CYP450s. These findings provide new insights into the recombinant expression of insect CYP450s in E. coli systems and will facilitate the theoretical approaches for functional expression and production of eukaryotic CYP450s.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
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Sistema Enzimático do Citocromo P-450
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Escherichia coli
Limite:
Animals
Idioma:
En
Revista:
Int J Biol Macromol
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
China