Affinity chromatography method expression

In this chapter, we describe our methods for purification of eIF2B holo-complexes as well as the catalytic subcomplex and the catalytic eIF2Bfi subunit (encoded by GCD6 in yeast) alone. We typically epitope tag only one eIF2B subunit, over-express the desired subunit combination, and use a single affinity chromatography step to purify the desired complex. In early work, a... 

Earlier work in this field [28] indicated that acetylcholinesterase enzymes would be suitable biomolecules for the purpose of pesticide detection, however, it was found that the sensitivity of the method varied with the type and source of cholinesterase used. Therefore the initial thrust of this work was the development of a range of enzymes via selective mutations of the Drosophila melanogaster acetylcholinesterase Dm. AChE. For example mutations of the (Dm. AChE) were made by site-directed mutagenesis expressed within baculovirus [29]. The acetylcholinesterases were then purified by affinity chromatography [30]. Different strategies were used to obtain these mutants, namely (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site,... 

The His-tagged MBP-MS2 coat fusion protein (HMM) used in this method was created to allow the protein to be immobilized on both Ni2+ or amylose affinity resins. HMM is a 59-kDa protein containing an NT-terminal hexahistidine (6x His) tag, a central maltose-binding protein (MBP) domain, and a C-terminal MS2 coat protein containing the V29/dIFG mutations, which prevent protein multimerization and increase its affinity for RNA (Lim and Peabody, 1994). The protein is expressed in E. coli from plasmid pHMM, which confers kanamycin resistance, and purified using affinity chromatography. 

Yan and his colleagues used a stable isotope labeled proteome internal standard to analyse protein expression alterations during oxidative stress in breast epithelial cells. Affinity chromatography was combined with MS/MS in the proteomic analysis of human 06-methylguanine-DNA methyltransferase. These studies demonstrated that proteomics can elucidate protein expre-ssions associated with the pharmacological and toxic effects of an anticancer drug when integrated with other biochemical methods. 

Wizemann H. and von Brunn A. 1999. Purification of E. co/i-expressed His-tagged hepatitis B core antigen by Ni -chelate affinity chromatography. J. Virol. Methods, 77, 189-197. 

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