Overexpression and purification of recombinant eRF1 proteins of rabbit and Tetrahymena thermophila.
The polypeptide release factor (eRF1) gene was cloned from rabbit and its overexpression and purification system was established in parallel with that of the eRF1 gene of Tetrahymena thermophila that has been cloned recently in this laboratory. The rabbit eRF1 (Ra-eRF1) is composed of 437 amino acids and is completely identical to human eRF1 though 3% distinct in the nucleotide sequence. This is in sharp contrast to Tetrahymena eRF1 (Tt-eRF1) that is only 57% identical to human eRF1. The recombinant Ra-eRF1 was marked with a histidine tag, overexpressed, and purified to homogeneity by two-step chromatography using Ni-NTA-agarose and Mono Q columns. In contrast to Ra-eRF1, Tt-eRF1 formed aggregates upon overexpression in Escherichia coli, hence it was purified under denaturing conditions, and used to raise rabbit antibody. The resulting anti-Tt-eRF1 antibody proved useful for examining conditions for soluble Tt-eRF1 in test cells. Finally, a soluble Tt-eRF1 fraction was purified from Saccharomyces cerevisiae transformed with the Tt-eRF1 expression plasmid by three steps of affinity and anion exchange chromatography. The cloned Ra-eRF1 gene complemented a temperature-sensitive allele in the eRF1 gene, sup45 (ts), of S. cerevisiae, though the complementation activity was significantly impaired by the histidine tag, whereas Tt-eRF1 failed to complement the sup45 (ts) allele.
Karamyshev
A L
AL
Department of Tumor Biology, Institute of Medical Science, University of Tokyo, Tokyo, 108-8630, Japan.
Karamysheva
Z N
ZN
Ito
K
K
Matsufuji
S
S
Nakamura
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005800:Genes, Fungal; D017125:Genes, Protozoan; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D010454:Peptide Termination Factors / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D016808:Tetrahymena thermophila / Q000235:genetics*
D000906:Antibodies; D017931:DNA Primers; D018076:DNA, Complementary; C489002:ETF1 protein, human; D010454:Peptide Termination Factors; D015800:Protozoan Proteins; D011994:Recombinant Proteins