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.

Capture of a monoclonal antibody and prediction of separation conditions using a synthetic multimodal ligand attached on chips and beads.

A synthetic ligand called 2-mercapto-5-benzimidazolesulfonic acid has been successfully used for the specific chromatographic capture of antibodies from a cell culture supernatant. Adsorption occurred at physiological ionic strength and pH range between 5.0 and 6.0, with some binding capacity variations within this pH range: antibody uptake increased when the pH decreased. With very dilute feedstocks, as was the case with the cell culture supernatant under investigation, it was found that the pH had to be slightly lowered to get a good antibody sorption capacity. To optimize separation conditions, a preliminary study was made using ProteinChip Arrays that displayed the same chemical functionalities as the resin. Arrays were analyzed using SELDI-MS. By this mean, it was possible to cross-over simultaneously different pH conditions at the adsorption and the desorption steps. Best conditions were implemented for preparative separation using regular lab-scale columns. At pH 5.2, antibody adsorption was not complete, while at pH 5.0 the antibody was entirely captured. pH 9 was selected at elution, rather than pH 8.0 or 10.0, and resulted in a complete desorption of antibodies from the column. Benefits of the prediction of separation conditions of antibodies on MBI beads using SELDI-MS were a significant reduction in analysis time and in sample volume. This was possible because the separation of IgG on the chip surface did mimic very well the separation on beads.

Repression of transcription at the human T-cell receptor Vbeta2.2 segment is mediated by a MAX/MAD/mSin3 complex acting as a scaffold for HDAC activity.

The identification of protein components in complex networks of co-regulators responsible for the modulation of proliferation versus differentiation modes of cell growth is a major problem. We use a combination of surface enhanced laser desorption/ionization mass spectrometry, surface plasmon resonance coupled to electrospray mass spectrometry, and immunoelectromobility shift assays to identify members of the MAX/MAD family binding to a specific DNA silencer fragment involved in the regulation of transcription for the human T-cell receptor Vbeta2.2 segment. We also identify the cofactors mSin3 and N-CoR known to interact with histone deacetylases. Inhibition of deacetylase activity in Jurkat cells prevented transcription inhibitor complex formation at the Vbeta2.2 segment, suggesting that this is either directly or indirectly dependent on the presence of HDACs.