Complex Purification Strategies

Expression of a recombinant protein using an inducible vector system would permit expression at endogenous levels to simulate physiologic levels of expression of a protein of interest. Tandem affinity purification strategies have recently been employed and facilitate the analyses of highly interactive proteins when the bait protein is expressed at endogenous levels. Immunoaffinity or immunoprecipitation followed by LC-MS/MS does not readily permit determination of the stoichiometry of interacting partners. Additionally, when compared to yeast hybrid experiments, it is difficult to determine whether interactions are binary when identified in complexes by MS/MS. 

Rubio, V., Shen, Y., Saijo, Y., Liu, Y., Gusmaroli, G., Dinesh-Ktrmar, S.P. and Deng, X.W. (2005) An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation. Plant J. 4J, 767-778. 

While the overall purification strategy for the recombinant mammalian Secl3/31 complex is relatively straightforward, there are a few caveats that... 

As a result, gradient fiactionation is a means of determining the subcellular localization of a protein antigen and whether it sediments as a component of a high molecular mass complex. Gradient fiactionation may also be usefiil as an emichment step in a purification strategy. 

As shown in the purification of rat histone H2A1 53 amide synthesized using the FMOC strategy, remarkable results using a Pharmacia Biotech Mono S column were obtained for the purification of complex synthetic mixtures (Figures 3 and 4)J5H] This system has been used with similar success with several other peptides. With a complex mixture of peptide/proteins, ion-exchange chromatography was found to be an important first step for preliminary fractionation to be further purified by RP-HPLC. 

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