In vivo incorporation of selenomethionine in proteins using Pseudomonas aeruginosa as expression host: case study-the outer membrane receptor FpvA.

Fiche publication


Date publication

novembre 2004

Auteurs

Membres identifiés du Cancéropôle Est :
Dr VAN DORSSELAER Alain


Tous les auteurs :
Folschweiller N, Pacaud K, Celia H, Potier N, Cobessi D, Van Dorsselaer A, Pattus F

Résumé

The number of protein structures solved using multiwavelength anomalous diffraction methods coupled with selenomethionine substitution has grown dramatically over the last years. We show using the outer membrane pyoverdin receptor FpvA that Pseudomonas aeruginosa can be used for producing proteins with a high level of selenomethionine incorporation. To circumvent problems encountered with mass spectroscopy analysis of purified membrane proteins, in-gel trypsin digestion of FpvA coupled with MALDI mass spectrometry analysis of the resulting peptides was used to determine the extent of selenomethionine incorporation. Selenomethionine incorporation greater than 95% was achieved using P. aeruginosa as an overexpression system.

Référence

Protein Expr Purif. 2004 Nov;38(1):79-83.