Purification and characterization of recombinant Xenopus poly(A)(+)-binding protein expressed in a baculovirus system.
The poly(A)(+)-binding protein (PABP) is a highly conserved protein that binds to the poly(A)+ tail of mRNAs. PABP has been shown to regulate message stability and translational efficiency, yet the mechanisms remain unknown. To facilitate further dissection of the functions of this protein, we have expressed and purified Xenopus PABP using a baculovirus expression system. At 48 h after infection of insect Spodoptera frugiperda (Sf9) cells with recombinant virus, approx. 3% of cell protein was PABP. Purification of PABP was achieved by affinity chromatography on poly(A)(+)-Sepharose. The purified protein was indistinguishable from Xenopus PABP with respect to its immunoreactivity and electrophoretic mobility. Furthermore, the recombinant PABP was expressed and purified as a functional protein as indicated by its ability to bind to poly(A)(+)-Sepharose and its ability to enhance the translation of adenylated messages in vitro. By comparing protein extracts from various developmental stages of Xenopus embryos with known amounts of purified PABP, we determined the amount of PABP per embryo. This analysis suggested that there is less than one PABP molecule available per PABP-binding site at early stages of development, and only a slight excess of PABP at later stages.
D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D005822:Genetic Vectors; D009036:Moths; D011061:Poly A / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014981:Xenopus
D002352:Carrier Proteins; D039102:Poly(A)-Binding Proteins; D011994:Recombinant Proteins; D011061:Poly A; D012313:RNA