In vivo incorporation of selenomethionine in proteins using Pseudomonas aeruginosa as expression host: case study-the outer membrane receptor FpvA.
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.
Folschweiller
Nicolas
N
Récepteurs et Protéines Membranaires, UPR CNRS 9050, BP10413, F-67412 Illkirch, France.
Pacaud
Karine
K
Celia
Hervé
H
Potier
Noëlle
N
Cobessi
David
D
Van Dorsselaer
Alain
A
Pattus
Franc
F
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D012645:Selenomethionine / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001425:Bacterial Outer Membrane Proteins; C082326:FpvA protein, Pseudomonas aeruginosa; D012645:Selenomethionine