Expression and Purification of Yeast Erf1 Mutant Proteins in Escherichia Coli

Fida Madayanti¹, Prima Endang Susilowati¹,³ Pingkan Aditiawati² and Akhmaloka¹*

¹Biochemistry Research Group, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jln Ganesha 10, Bandung Indonesia.

²School of Life Science and Technology, Institut Teknologi Bandung, Jln Ganesha 10, Bandung Indonesia.

³Departement of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Haluoleo, Kendari Indonesia.

ABSTRACT: Translation termination in eukaryotes is mediated by two release factors, eRF1 and eRF3, which interact to form a heterodimer that mediates termination at all three stop codons. Detailed mechanism of the interaction between the two proteins, however, was still unclear yet. Previous studies indicated that threonin-295 on the third domain of eRF1, involved in its interaction to eRF3. In other to further characterize the role of threonine at position 295 on its interaction, two SUP45 mutants, namely sup45-T295A and sup45-T295S, were constructed and expressed in Escherichia coli. The mutations were successfully performed by PCR megaprimer and confirmed by sequence analysis. The mutant genes were over expressed in Escherichia coli BL21(DE3) under the promotor of T7 using pUKC630 vector. The mutant proteins namely, eRF1-T295A and eRF1-T295S, were expressed over 19% and 18% of total protein, respectively. The proteins were successfully purified by one step purification through Immobilized Metal Affinity Chromatography (IMAC).

KEYWORDS: ERF1; sup45 mutants; over-expression; IMAC purification

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