Generation of human steroidogenic cytochrome P450 enzymes for structural and functional characterization.
Methods Enzymol
; 689: 3-38, 2023.
Article
em En
| MEDLINE
| ID: mdl-37802575
ABSTRACT
Six cytochrome P450 enzymes are involved in human steroidogenesis, converting cholesterol to sex steroids, mineralocorticoids, and glucocorticoids. While early work was accomplished with steroidogenic P450 orthologs from more accessible sources, knowledge of basic biochemistry through successful drug design have been greatly facilitated by recombinantly-expressed, highly purified human versions of these membrane proteins. Many membrane proteins are difficult to express and purify and are unstable. Membrane P450 expression in E. coli has been facilitated by modification and/or truncation of the membrane-interacting N-terminus, while metal-affinity resins and histidine-tagging greatly facilitates purification. However, substantial optimization is still frequently required to maintain protein stability. Over time, a generalized three-column purification scheme has been developed and tweaked to generate substantial quantities of fully active, highly purified human cytochrome P450 enzymes that have made possible the application of many structural, biochemical, and biophysical techniques to elucidate the mysteries of these critical human enzymes.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sistema Enzimático do Citocromo P-450
/
Escherichia coli
Limite:
Humans
Idioma:
En
Revista:
Methods Enzymol
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos