High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics
| Autor: | Paul G. Blommel, Frank C. Vojtik, Grzegorz Sabat, George N. Phillips, Craig A. Bingman, Holalkere V. Geetha, Janet E. McCombs, Brendan T. Burns, Hassan K. Sreenath, Amy C. Harms, Andrew C. Olson, Michael R. Sussman, Brian G. Fox, Jason M. Ellefson, David J. Aceti, Won Bae Jeon, Kory D. Seder |
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| Rok vydání: | 2005 |
| Předmět: |
Proteomics
Quality Control Spectrometry Mass Electrospray Ionization Electrospray ionization Arabidopsis Biology Mass spectrometry medicine.disease_cause Biochemistry Chromatography Affinity Structural genomics law.invention Structural Biology law Genetics medicine TEV protease Escherichia coli Selenomethionine Carbon Isotopes Chromatography Nitrogen Isotopes Arabidopsis Proteins General Medicine Fusion protein Recombinant Proteins Matrix-assisted laser desorption/ionization Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Recombinant DNA |
| Zdroj: | Journal of structural and functional genomics. 6(2-3) |
| ISSN: | 1345-711X |
| Popis: | The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR. |
| Databáze: | OpenAIRE |
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