Rapid high-yield expression and purification of fully post-translationally modified recombinant clusterin and mutants.
Sci Rep
; 10(1): 14243, 2020 08 28.
Article
en En
| MEDLINE
| ID: mdl-32859921
ABSTRACT
The first described and best known mammalian secreted chaperone, abundant in human blood, is clusterin. Recent independent studies are now exploring the potential use of clusterin as a therapeutic in a variety of disease contexts. In the past, the extensive post-translational processing of clusterin, coupled with its potent binding to essentially any misfolded protein, have meant that its expression as a fully functional recombinant protein has been very difficult. We report here the first rapid and high-yield system for the expression and purification of fully post-translationally modified and chaperone-active clusterin. Only 5-6 days is required from initial transfection to harvest of the protein-free culture medium containing the recombinant product. Purification to near-homogeneity can then be accomplished in a single affinity purification step and the yield for wild type human clusterin is of the order of 30-40 mg per litre of culture. We have also shown that this system can be used to quickly express and purify custom-designed clusterin mutants. These advances dramatically increase the feasibility of detailed structure-function analysis of the clusterin molecule and will facilitate identification of those specific regions responsible for the interactions of clusterin with receptors and other molecules.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Ingeniería de Proteínas
/
Clusterina
Idioma:
En
Revista:
Sci Rep
Año:
2020
Tipo del documento:
Article
País de afiliación:
Australia