RRC ID 534
Author Abo Y, Hagiya A, Naganuma T, Tohkairin Y, Shiomi K, Kajiura Z, Hachimori A, Uchiumi T, Nakagaki M.
Title Baculovirus-mediated expression and isolation of human ribosomal phosphoprotein P0 carrying a GST-tag in a functional state.
Journal Biochem. Biophys. Res. Commun.
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.
Volume 322(3)
Pages 814-9
Published 2004-9-24
DOI 10.1016/j.bbrc.2004.07.196
PII S0006-291X(04)01728-0
PMID 15336536
MeSH Animals Baculoviridae / genetics* Cell Line Gene Expression Regulation, Viral / genetics Genetic Vectors Glutathione Transferase / genetics Humans Phosphoproteins / genetics* Phosphoproteins / isolation & purification Phosphoproteins / metabolism Recombinant Fusion Proteins / isolation & purification Recombinant Fusion Proteins / metabolism Ribosomal Proteins / genetics* Ribosomal Proteins / isolation & purification Ribosomal Proteins / metabolism Spodoptera Transfection
IF 2.559
Times Cited 1
WOS Category BIOPHYSICS BIOCHEMISTRY & MOLECULAR BIOLOGY
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