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.

ABSTRACT: 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 a

SUBMITTER: Cao H 

PROVIDER: S-EPMC1351391 | biostudies-literature | 2003 Apr

REPOSITORIES: biostudies-literature