Overexpression and Purification of Recombinant eRF1 Proteins of Rabbit and Tetrahymena thermophila

Abstract

Termination of translation during protein biosynthesis takes place on the ribosome in response to a stop, rather than a sense, codon in the decoding site. This process requires two classes of polypeptide release factors (RFs): a class I factor, codon-specific RF (RF1 and RF2 in prokaryotes; eRF1 in eukaryotes), and a class II factor, nonspecific RF (RF3 in prokaryotes; eRF3 in eukaryotes) that binds guanine nucleotides and stimulates class I RF activity Although the termination process was first discovered in the late 1960s, much of the mechanism has remained obscure. One of the reasons for relatively slow progresses in the study of translation termination mechanisms was the lack of a complete in vitro system that catalyzes initiation, elongation, and termination of protein synthesis equivalent to the in vivo process. The classical, but still useful, in vitro system of translation termination was to monitor the rate of N-formyl-methionine (fMet) release from the ribosomefMettRNA complex at the stop codon In this study, we cloned the rabbit eRF1 gene to facilitate the in vitro translation system composed of rabbit reticulocyte lysate under homologous conditions. One of the assay systems that we are engaged in the construction of is to measure competition between translation termination and frameshifting using the mammalian antizyme frameshift construct of mRNA (unpublished). Cloning and purification of rabbit eRF1, designated Ra-eRF1, is along this line. 0006-2979/99/6412-1391$22.00 ©2000 ÌÀÈÊ Íàóêà/Interperiodica * To whom correspondence should be addressed. Vol. 64, No. 12, 1999, pp. 1391-1400. Translated from Biokhimiya, Vol. 64, No. 12, 1999, pp. 1648-1658. Original Russian Text Copyright © 1999 AbstractThe 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, TteRF1 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