Green, S.R. and Spalding, A. and Ashford, T. and Proud, C.G. and Tuite, M.F. (1991) Synthesis of human initiation factor-2-alpha in saccharomyces-cerevisiae. Gene, 108 (2). pp. 253-258. ISSN 0378-1119.
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A human eIF-2-alpha cDNA (encoding alpha-subunit of the eukaryotic initiation factor-2) was expressed under the control of the galactose-regulated GAL1,10 promoter, in Saccharomyces cerevisiae, in order to study the possible interactions of human eIF-2-alpha with the yeast protein synthesis apparatus. Isoelectric focusing coupled with Western-blot analysis demonstrated that the human eIF-2-alpha subunit synthesized in yeast under a variety of growth conditions was detected as two bands which co-migrated with the phosphorylated and unphosphorylated forms of rabbit eIF-2-alpha, suggesting covalent modification in vivo. Cell fractionation studies further demonstrated that the synthesised human eIF-2-alpha protein, though present in the cytoplasm, was largely associated with the yeast ribosomes, but could be removed from these by washing with 0.3 M KCl. This possible association of the synthesised human subunit into a three-subunit (alpha, beta and gamma) eIF-2 complex was further examined by partial purification of the yeast eIF-2 complex and estimation of the molecular mass of this complex. Immunoreactive eIF-2-alpha was found in fractions with eIF-2 activity and the estimated molecular mass (130 kDa) corresponded to that predicted for the eIF-2 trimer. These analyses suggest that human eIF-2-alpha subunit synthesised in yeast can become involved with the yeast protein synthetic apparatus, though whether this is a functional incorporation requires further genetic studies.
|Uncontrolled keywords:||recombinant dna; yeast; translation factor; eif-2-alpha; heterologous gene expression|
|Subjects:||Q Science > QH Natural history > QH426 Genetics|
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences|
|Depositing User:||O.O. Odanye|
|Date Deposited:||09 Oct 2009 09:56|
|Last Modified:||09 Oct 2009 09:56|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/22959 (The current URI for this page, for reference purposes)|
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