An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins.
The recombinant expression of integral membrane proteins is considered a major challenge, and together with the crystallization step, the major hurdle toward routine structure determination of membrane proteins. Basic methodologies for high-throughput (HTP) expression optimization of soluble proteins have recently emerged, providing statistically significant success rates for producing such proteins. Experimental procedures for handling integral membrane proteins are generally more challenging, and there have been no previous comprehensive reports of HTP technology for membrane protein production. Here, we present a generic and integrated parallel HTP strategy for cloning and expression screening of membrane proteins in their detergent solubilized form. Based on this strategy, we provide overall success rates for membrane protein production in Escherichia coli, as well as initial benchmarking statistics of parameters such as expression vectors, strains, and solubilizing detergents. The technologies were applied to 49 E. coli integral membrane proteins with human homologs and revealed that 71% of these proteins could be produced at sufficient levels to allow milligram amounts of protein to be relatively easily purified, which is a significantly higher success rate than anticipated. We attribute the high success rate to the quality and robustness of the methodology used, and to introducing multiple parameters such as different vectors, strains, and detergents. The presented strategy demonstrates the usefulness of HTP technologies for membrane protein production, and the feasibility of large-scale programs for elucidation of structure and function of bacterial integral membrane proteins.
Eshaghi
Said
S
Department of Biochemistry and Biophysics, Stockholm University, Roslagstullsbacken 15, Albanova University Center, SE-114 21 Stockholm, Sweden.
Hedrén
Marie
M
Nasser
Marina Ignatushchenko Abdel
MI
Hammarberg
Tove
T
Thornell
Anders
A
Nordlund
Pär
P
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D003902:Detergents; D008565:Membrane Proteins; D011994:Recombinant Proteins