Protein Purification

Requirements:

Eukaryotic Initiation Factor 2 (eIF2) is a heterotrimeric (a, b, g ) protein complex that delivers the Met-tRNA i to the AUG start codon during translational initiation. The left panel depicts an SDS-PAGE of purified eIF2 from three different yeast strains that express His 8­ tagged versions of a wild-type, a Y142H mutation, or a K250R mutation of the g subunit. Crude extracts were purified through a Ni +2 -Agarose column followed by a Heparin column. The right panel depicts an equilibrium binding assay of Met-tRNA i to the various purified eIF2 complexes. (adapted from Erickson and Hannig. EMBO. 1996;15(22):6311-6320.) Which step in the purification procedure removed contaminating proteins that may contain structural motifs that are similar to a His 8 tag? What is the estimated purity of the wild-type eIF2? What is the estimated MW of eIF2g? What is the estimated MW of eIF2? Based upon the data, what specific role does the amino acids K250 and Y142 in eIF2g function?

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