Generation of human steroidogenic cytochrome P450 enzymes for structural and functional characterization.
Methods Enzymol
; 689: 3-38, 2023.
Article
in 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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Cytochrome P-450 Enzyme System
/
Escherichia coli
Limits:
Humans
Language:
En
Journal:
Methods Enzymol
Year:
2023
Document type:
Article
Affiliation country:
United States