Optimising the transient expression of GABA(A) receptors in adherent HEK293 cells.
Protein Expr Purif
; 154: 7-15, 2019 02.
Article
in En
| MEDLINE
| ID: mdl-30248449
ABSTRACT
Owing to their therapeutic relevance, considerable efforts are devoted to the structural characterisation of membrane proteins. Such studies are limited by the availability of high quality protein due to the difficulty of overexpression in recombinant mammalian systems. We sought to systematically optimise multiple aspects in the process of transiently transfecting HEK293â¯cells, to allow the rapid expression of membrane proteins, without the lengthy process of stable clone formation. We assessed the impact of medium formulation, cell line, and harvest time on the expression of GABAA receptors, as determined by [3H]muscimol binding in cell membranes. Furthermore, transfection with the use of calcium phosphate/polyethyleneimine multishell nanoparticles was optimised, and a dual vector system utilising viral enhancing elements was designed and implemented. These efforts resulted in a 40-fold improvement in GABAA α1ß3 receptor expression, providing final yields of 22â¯fmol/cm2. The findings from this work provide a guide to the optimisation of transient expression of proteins in mammalian cells and should assist in the structural characterisation of membrane proteins.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Transfection
/
Gene Expression
/
Cell Membrane
/
Receptors, GABA-A
Limits:
Humans
Language:
En
Journal:
Protein Expr Purif
Journal subject:
BIOLOGIA MOLECULAR
Year:
2019
Document type:
Article
Affiliation country:
Australia