Purification of the large ribosomal subunit via its association with the small subunit.
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.
Simons
Samuel P
SP
Department of Exploratory Medicinal Sciences, Pfizer Global Research and Development, Groton, CT 06340, USA.
McLellan
Thomas J
TJ
Aeed
Paul A
PA
Zaniewski
Richard P
RP
Desbonnet
Charlene R
CR
Wondrack
Lillian M
LM
Marr
Eric S
ES
Subashi
Timothy A
TA
Dougherty
Thomas J
TJ
Xu
Zuoyu
Z
Wang
Ing-Kae
IK
LeMotte
Peter K
PK
Maguire
Bruce A
BA
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
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