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doi:10.1016/j.pep.2006.02.021 
Copyright © 2006 Elsevier Inc. All rights reserved.
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
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Virginie Brenaca, 
, 
, Nicolas Mouzb, Anthony Schapmana and Vincent Ravaulta
Received 18 October 2005;
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® arrays. The highest level of expression was found in Rosetta (DE3) with a C-terminal construct after induction at 37 °C. The purification scheme was elucidated using SELDI-MS: S-LAT was efficiently captured on an IMAC ProteinChip array saturated with nickel ions (Ni2+) and then fractionated on a Q ProteinChip array. These conditions were directly transferred to IMAC-Ni2+ 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.
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
Article Outline
- Materials and methods
- Chemicals, biologicals, and equipment
- Construction of expression vectors
- Protein expression and cell lysis
- NP20 array preparation
- IMAC30 array preparation
- CM10 and Q10 array preparation
- H50 array preparation
- Sinapinic acid addition and array reading
- Sequential LC runs using micro-volumes of IMAC and of Q chromatography sorbents
- SDS–PAGE analysis and in-gel trypsin digest
- Results and discussion
- Selection of the expression strategy of S-LAT using IMAC ProteinChip arrays
- S-LAT purification process development using ProteinChip arrays
- Partial purification of S-LAT using micro-volumes of chromatography sorbents
- Peptide mapping after in-gel trypsin digest of partially purified S-LAT
- Conclusion
- Acknowledgements
- References
