Expression and purification of recombinant wheat translation initiation factor eIF2

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Expression and purification of recombinant wheat translation initiation factor eIF2

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Title: Expression and purification of recombinant wheat translation initiation factor eIF2
Author: Tzeng, Shinetin
Abstract: Proteins carry out the activities for cellular growth and maintenance in a timely and space-specific manner, so the formation of proteins from their genetic counterparts, or translation, must be tightly controlled. Regulation occurs primarily during the initiation step of translation, a highly involved process that properly positions messenger RNA, methionine-charged initiator transfer RNA, and ribosomes via a family of proteins known as eukaryotic initiation factors (eIFs). One of these proteins is eIF2, a trimeric complex made of eIF2α, eIF2β, and eIF2γ. In mammalian and yeast systems, the phosphorylation of eIF2α by specific kinases and its downstream effects down-regulate global protein synthesis, and the mechanism has been widely studied. However, in plants, evidence suggests that different regulatory pathways may be involved. To ultimately describe the molecular and biochemical basis by which higher plants regulate protein synthesis, studies of mutant eIF2α and the effects of phosphorylation are necessary. To achieve this goal, genes for the three subunits of wheat eIF2 were synthesized de novo by overlapping oligonucleotides that were optimized for E. coli codon usage, and then the genes were cloned into an operon for expression of the complex in E. coli. This particular study focuses on optimizing the conditions for expression and purification of soluble eIF2 complexes. Alterations in the strain of E. coli, growth condition and media, and operon construct were tested to enhance eIF2 expression and solubility, and different methods of purification were applied. However, at this point, the expression and purification of eIF2 still remains unsuccessful.
Department: Biological Sciences, School of
Subject: College of Natural Sciences translation proteins eIF2 wheat purification
URI: http://hdl.handle.net/2152/13466
Date: 2010

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