Expression optimization and purification process development of an engineered soluble recombinant mouse linker of activation of T cells using surface enhanced laser desorption/ionization-mass spectrometry.
Protein purification development is the bottleneck of recombinant protein production therefore there is a need to shorten process development and monitoring. Surface enhanced laser desorption/ionization-mass spectrometry (SELDI-MS) was evaluated to optimize the expression and to develop the purification of a recombinant mouse protein: a transmembrane adaptor involved in T cell receptor signaling named "linker for activation of T cells" (LAT). The protein was expressed as a soluble form (S-LAT) in three strains of Escherichia coli: BL21 (DE3), Rosetta (DE3), and BL21 (DE3) pLys S. The expression of S-LAT was monitored on immobilized metal affinity chromatography (IMAC) ProteinChip arrays. The highest level of expression was found in Rosetta (DE3) with a C-terminal construct after induction at 37 degrees C. The purification scheme was elucidated using SELDI-MS: S-LAT was efficiently captured on an IMAC ProteinChip array saturated with nickel ions (Ni(2+)) and then fractionated on a Q ProteinChip array. These conditions were directly transferred to IMAC-Ni(2+) HyperCel and Q Ceramic HyperD F chromatography sorbents. After these two purification steps, S-LAT was estimated to be more than 80% pure, confirming a very good match between array and sorbent. Finally, a peptide mapping was performed on a hydrophobic array after in gel trypsin digest, verifying that the purified protein was the mouse LAT. This is the first report of a protocol for the production and purification of S-LAT. The selection of the best expression and purification strategy along with the identification were enabled in 5 days with less than 5 mL of soluble fraction of crude culture samples.
Brenac
Virginie
V
Pall BioSepra, Cergy St. Christophe, France. vbrenac@europe.pall.com
Mouz
Nicolas
N
Schapman
Anthony
A
Ravault
Vincent
V
D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D002853:Chromatography, Liquid; D004926:Escherichia coli; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D051379:Mice; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D040081:Protein Array Analysis; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D048868:Adaptor Proteins, Signal Transducing; C491734:Lat protein, mouse; D008565:Membrane Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins