Enhancement of the StreptoTag method for isolation of endogenously expressed proteins with complex RNA binding targets.
StreptoTag is a novel affinity chromatography-based method for the isolation of high- and low-affinity RNA binding proteins. Originally it was shown possible to isolate recombinant protein from yeast or bacterial extracts using small, specific, well-characterised RNA binding targets. Here we show that using an enhanced aptamer it is not only possible to efficiently immobilise large, highly structured RNA binding targets onto the streptomycin columns but also that the StreptoTag method can be used for the isolation and purification of endogenously expressed regulatory proteins, with relatively low abundance, from eukaryotic extracts. As an example for this we uncover the identity of a karyophilic cellular protein which specifically binds to an area within the large, highly folded structure that characterises the mRNA from the unique 3' region (U3) of the mouse mammary tumour virus (MMTV) long terminal repeat (LTR). Hence, this method is now suitable for the quick and efficient isolation and identification of novel RNA binding proteins such as regulatory factors.
Dangerfield
John A
JA
Christian Doppler Laboratory for Gene Therapeutic Vectors, Vienna, Austria. john.dangerfield@vu-wien.ac.at
Windbichler
Nikolai
N
Salmons
Brian
B
Günzburg
Walter H
WH
Schröder
Renée
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D004268:DNA-Binding Proteins; D006801:Humans; D066298:In Vitro Techniques; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072260:RNA Splicing Factors; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012097:Repressor Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013307:Streptomycin; D020079:Terminal Repeat Sequences
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C410595:poly-U binding splicing factor 60KDa; D012313:RNA; D013307:Streptomycin