title: Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state.
abstract: We constructed an overexpression system for human ribosomal phosphoprotein P0, together with P1 and P2, which is crucially important for translation. Genes for these proteins, fused with the glutathione S-transferase (GST)-tag at the N-terminus, were inserted into baculovirus and introduced to insect cells. The fusion proteins, but not the proteins without the tag, were efficiently expressed into cells as soluble forms. The fusion protein GST.P0 as well as GST.P1/GST.P2 was phosphorylated in cells as detected by incorporation of (32)P and reactivity with monoclonal anti-phosphoserine antibody. GST.P0 expressed in insect cells, but not the protein obtained in Escherichia coli, had the ability to form a complex with P1 and P2 proteins and to bind to 28S rRNA. Moreover, the GST.P0-P1-P2 complex participated in high eEF-2-dependent GTPase activity. Baculovirus expression systems appear to provide recombinant human P0 samples that can be used for studies on the structure and function.
authors.affiliation: Laboratory of Silkworm Genetics and Pathology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan.
authors.forename: Yohichi
authors.initials: Y
authors.lastname: Abo
authors.forename: Akiko
authors.initials: A
authors.lastname: Hagiya
authors.forename: Takao
authors.initials: T
authors.lastname: Naganuma
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Tohkairin
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Shiomi
authors.forename: Zenta
authors.initials: Z
authors.lastname: Kajiura
authors.forename: Akira
authors.initials: A
authors.lastname: Hachimori
authors.forename: Toshio
authors.initials: T
authors.lastname: Uchiumi
authors.forename: Masao
authors.initials: M
authors.lastname: Nakagaki
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D010750:Phosphoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
chemical_list: D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C054675:ribosomal protein P0; D005982:Glutathione Transferase