title: Purification of the large ribosomal subunit via its association with the small subunit.
abstract: We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.
authors: Department of Exploratory Medicinal Sciences, Pfizer Global Research and Development, Groton, CT 06340, USA.
authors: Samuel P
authors: SP
authors: Simons
authors: Thomas J
authors: TJ
authors: McLellan
authors: Paul A
authors: PA
authors: Aeed
authors: Richard P
authors: RP
authors: Zaniewski
authors: Charlene R
authors: CR
authors: Desbonnet
authors: Lillian M
authors: LM
authors: Wondrack
authors: Eric S
authors: ES
authors: Marr
authors: Timothy A
authors: TA
authors: Subashi
authors: Thomas J
authors: TJ
authors: Dougherty
authors: Zuoyu
authors: Z
authors: Xu
authors: Ing-Kae
authors: IK
authors: Wang
authors: Peter K
authors: PK
authors: LeMotte
authors: Bruce A
authors: BA
authors: Maguire
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D030562:Databases, Protein; D034301:Deinococcus / Q000648:ultrastructure*; D015870:Gene Expression; D015636:Magnesium Chloride; D009842:Oligopeptides; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000235:genetics; D012329:RNA, Bacterial / Q000032:analysis; D012335:RNA, Ribosomal / Q000032:analysis; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins / Q000032:analysis / Q000235:genetics; D054681:Ribosome Subunits, Large, Bacterial / Q000737:chemistry* / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C023509:ribosomal protein S16; D015636:Magnesium Chloride; C085706:FLAG peptide