Summary
High performance liquid chromatography was used to separate proteins derived from ribosomes of the archaebacteriumMethanococcus vannielii. Several methods of separation were tested: size exclusion chromatography, reversed phase and ion-exchange chromatography. Best results were obtained using reversed phase columns and volatile buffers that allow direct sequence analysis of the proteins.
In addition, HPLC was used to separate crosslinked protein-protein pairs fromBacillus stearothermophilus ribosomes on a preparative scale. Identification of crosslinked amino acids is a prerequisite for more detailed topographical investigations of this cell organelle. The purification of the 50S crosslink L23–L29 was achieved by a combination of two different reversed phase columns, and the 30S crosslink S13–S19 was purified after salt extraction by size exclusion chromatography.
The methods described here allow a rapid preparation of ribosomal proteins for structural and functional investigations.
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Abbreviations
- HPLC:
-
High Performance Liquid Chromatography
- M. vannielii :
-
Methanococcus vannielii
- B. stearothermophilus :
-
Bacillus stearothermophilus
- 2D:
-
two-dimensional
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Kamp, R.M., Brockmöller, J. & Wittmann-Liebold, B. Separation of ribosomal protein-protein crosslinks fromBacillus stearothermophilus and ribosomal proteins from archaebacteria by high performance liquid chromatography. Chromatographia 22, 249–254 (1986). https://doi.org/10.1007/BF02268768
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DOI: https://doi.org/10.1007/BF02268768