Expression and purification of recombinant tristetraprolin that can bind to tumor necrosis factor-alpha mRNA and serve as a substrate for mitogen-activated protein kinases.
Tristetraprolin (TTP) is an mRNA-binding protein, but studies of this interaction have been difficult due to problems with the purification of recombinant TTP. In the present study, we expressed human and mouse TTP as glutathione S-transferase and maltose-binding protein (MBP) fusion proteins in Escherichia coli, and purified them by affinity resins and Mono Q chromatography. TTP cleaved from the fusion protein was identified by immunoblotting, MALDI-MS, and protein sequencing, and was further purified to homogeneity by continuous-elution SDS-gel electrophoresis. Purified recombinant TTP bound to the AU-rich element of tumor necrosis factor-alpha (TNFalpha) mRNA and this binding was dependent on Zn(2+). Results from sizing columns suggested that the active species might be in the form of an oligomer of MBP-TTP. Recombinant TTP was phosphorylated by three members of the mitogen-activated protein (MAP) kinase family, p42, p38, and JNK, with half-maximal phosphorylation occurring at approximately 0.5, 0.25, and 0.25 microM protein, respectively. Phosphorylation by these kinases did not appear to affect the ability of TTP to bind to TNFalpha mRNA under the assay conditions. This study describes a procedure for purifying nonfusion protein TTP to homogeneity, demonstrates that TTP's RNA-binding activity is zinc dependent, and that TTP can be phosphorylated by JNK as well as by the other members of the greater MAP kinase family.
Cao
Heping
H
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Mail Drop F3-04, Research Triangle Park, NC 27709, USA. cao2@niehs.nih.gov
Dzineku
Frederick
F
Blackshear
Perry J
PJ
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D015151:Immunoblotting; D020935:MAP Kinase Signaling System; D058619:Maltose-Binding Proteins; D051379:Mice; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D013997:Time Factors; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D058619:Maltose-Binding Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D051816:Tristetraprolin; D014409:Tumor Necrosis Factor-alpha; C497552:ZFP36 protein, human; C497553:Zfp36 protein, mouse; D012313:RNA; D005982:Glutathione Transferase