부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.pep.2006.02.021 Byline: Virginie Brenac (a), Nicolas Mouz (b), Anthony Schapman (a), Vincent Ravault (a) Keywords: Soluble linker of activation of T cells; His-tagged recombinant protein; Surface enhanced laser desorption/ionization-mass spectrometry; Protein microarray; Immobilized metal affinity chromatography Abstract: 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.sup.[R] 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.sup.2+) and then fractionated on a Q ProteinChip array. These conditions were directly transferred to IMAC-Ni.sup.2+ HyperCel and Q Ceramic HyperD.sup.[R] 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 5mL of soluble fraction of crude culture samples. Author Affiliation: (a) Pall BioSepra, 48, avenue des Genottes, 95800 Cergy St. Christophe, France (b) Protein'eXpert, 15, rue des Martyrs, 38054 Grenoble, France Article History: Received 18 October 2005; Revised 31 January 2006