Overexpression and purification of recombinant eRF1 proteins of rabbit andTetrahymena thermophila

The polypeptide release factor (eRF1) gene was cloned from rabbit and its o verexpression 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 acid s and is completely identical to human eRF1 though 3% distinct in the nucle otide sequence. This is in sharp contrast to Tetrahymena eRF1 (Tt-eRF1) tha t is only 57% identical to human eRF1. The recombinant Ra-eRF1 was marked w ith 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, he nce it was purified under denaturing conditions, and used to raise rabbit a ntibody. The resulting anti-Tt-eRF1 antibody proved useful for examining co nditions for soluble Tt-eRF1 in test cells. Finally, a soluble Tt-eRF1 frac tion was purified from Saccharomyces cerevisiae transformed with the Tt-eRF 1 expression plasmid by three steps of affinity and anion exchange chromato graphy. 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.