Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state.
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.
Abo
Yohichi
Y
Laboratory of Silkworm Genetics and Pathology, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan.
Hagiya
Akiko
A
Naganuma
Takao
T
Tohkairin
Yukiko
Y
Shiomi
Kunihiro
K
Kajiura
Zenta
Z
Hachimori
Akira
A
Uchiumi
Toshio
T
Nakagaki
Masao
M
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
D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C054675:ribosomal protein P0; D005982:Glutathione Transferase