Systematic, computer-assisted optimisation of the isolation of Thermus thermophilus 50S ribosomal proteins by reversed-phase high-performance liquid chromatography.
Isolation of the 50S ribosomal proteins from Thermus thermophilus has been achieved for the first time using reversed-phase high-performance liquid chromatography based on the use of the non-end-capped LiChrospher RP-18 sorbent and computer-assisted method development for optimisation of the resolution. The separation approach for these basic ribosomal proteins utilised mobile phases of high ionic strength, to suppress silanophilic interactions with this type of reversed-phase sorbent. These conditions were found to be a key requirement for achieving good resolution with minimal peak-tailing. The retention times of the 50S ribosomal proteins of Thermus thermophilus were observed to be in very close agreement with the values predicted by computer simulation procedures based on linear solvent strength concepts, with an average error of only 0.5%, whilst base-line resolution was achieved for most of the adjacent peak zones. Following N-terminal sequencing, the proteins TthL5, TthL9, TthL18, TthL24, TthL29, TthL32, TthL34, TthL35 and TthL36 of Thermus thermophilus were readily identified. This approach thus provided a readily optimised strategy for the isolation of the 50S ribosomal proteins from Thermus thermophilus and should be generally applicable to the corresponding ribosomal proteins from various other species, as well as other classes of basic proteins present in crude extracts derived from other biological sources.
Boysen
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RI
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
Erdmann
V A
VA
Hearn
M T
MT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D003201:Computers; D015180:Electrophoresis, Gel, Two-Dimensional; D005069:Evaluation Studies as Topic; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D012269:Ribosomal Proteins; C035078:ribosomal protein L29