Protein complexes, purification

Puig O, Caspary F, Rigaut G, et al. The tandem affinity purification (TAP) method a general procedure of protein complex purification. Methods 2001 24 218-229. 

A major promise of this approach is that protein complex purification protocols can be simplified and mild washing conditions used thus, the ability to screen for transient or weak interaction partners is improved. This has been demonstrated by Aebersold and coworkers who used ICAT technology combined with MS analysis to identify specific interacting partners of a large RNA polymerase 11 (Pol 11) preinitiation complex (PIC) only partially purified from nuclear extracts by a single-step promoter DNA affinity procedure (Ranish et al., 2003). 

Puig, 0., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M. and Seraphin, B. (2001) The tandem affinity purification (TAP) method a general procedure of protein complex purification. Methods 24, 218-229. 

Fawley MW, Morton IS, Steward KD and Mattox KR (1987) Evidence fora common evolutionary origin of hght-harvesting fucoxanthin chlorophyll a/c protein complexes of Pavlova gyrans (Prymnesiophyceae) and Phaeodactylum tricomutum (Bacillariophyceae). J Phycol 23 377-381 Friedman AL and Alberte RS (1984) A diatom light-harvesting pigment protein complex Purification and characterization. Plant Physiol 76 483 89... 

Fig. 1 Schematics of tandem affinity purification (TAP). The methodology of using TAP technique for protein complex purification is outlined in 6 steps. (1) The target protein (the bait ) is co-transcribed with the tandem-arranged affinity tags to either the N- or the C-terminus of the gene (shown is the N-terminal fusion). The two affinity tags (shaded boxes 1 and 2j are separated by a protease cleavage site (blank bo)(j, from where the far-end tag would be cut off and the near-end one would be exposed to the affinity columns for a second-round purification. (2) The bait and the tags are expressed as a chimeric fusion protein. The bait would form a complex with its in vivo partners (depicted as A, B, and Q. Nonspecific association may happen as well, as depicted by D, E, and F. (3) The first round purification via specific interaction between the affinity binder 1 and the far-end tag eliminates most nonspecific associated proteins. (4) In the second round purification, the near-end tag is cut off and the protein complex is further purified, with most nonspecific associates removed. (5 and 6) The bait protein and its associated proteins are eluted from the column and are further analyzed either by using antibodies, or by mass spectrometry...

Bonk, M. et al.. Purification and characterization of chaperonin 60 and heat-shock protein 70 from chromoplast of Narcissus pseudonarcissus involvement of heat-shock protein 70 in a soluble protein complex containing phytoene desaturase. Plant Physiol. Ill, 931, 1996. 

Rigaut G, Shevchenko A, Rutz B, et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 1999 17 1030-1032. 

Further characterization of the dendrimer-coupled protein complexes was studied by hydrophobic interaction chromatography carried out by purification over an octyl-Sepharose column. The products obtained by reaction of SIAB with ALP and its complex with third-generation dendrimer, ALP-E3, were... 

In addition to the direct absorbance methods, colorimetric methods are suited for relatively pure proteins as purification progresses. They are accurate if calibrated from a standard curve of the test protein reference sample and fast if automated. However, they are not as simple to perform as direct absorbance methods. Hence they are not as suitable for production as direct absorbance methods. The relative simplicity of colorimetric methods makes them more suited to automated formulation and stability studies and total-protein assays of complex mixtures. Microtiter plate versions of colorimetric assays allow for automation and consumption of relatively small sample sizes while requiring little specialized equipment or training. 

As an example for combination of different methods in protein purification Fig. 3.2 gives the flow chart for the isolation of membrane protein complex. 

Matsumura, K., Nagano, M., and Fusetani, N., Purification of a larval settlement-inducing protein complex (SIPC) of the barnacle Balanus amphitrite, J. Exp. Zool., 281, 12, 1998. 

Alterations of the mitochondrial membrane may precede those that occur in the nucleus. These changes can involve both the inner and the outer membrane, leading to a dissipation of the transmembrane potential and/or to the release of intermembrane proteins through the outer membrane. The main group of proteins responsible for mitochondrial alterations consist of the proteins known as Bax, which form pores in the outer membrane, causing the release of cytochrome c to the cytoplasm (Loeffler and Kroemer, 2000 Arden and Betenbaugh, 2004). Western blot techniques can be used to specifically detect the presence of cytochrome c in the cytoplasm of apoptotic cells. However, complex purification protocols are required, and there is the possibility of incomplete separation of mitochondria from the cytoplasm therefore, this technique is not very popular. 

There was a second very good reason for doing this work at the present time—it has recently become possible This is because of the advances in isolation, purification and crystallisation of the key pigment-protein complexes of photosynthetic organisms as well as the improving time resolution and precision of time-resolved spectroscopic techniques in the picosecond and sub-picosecond region. 

Figure 4.8. MuDPIT platform. The MuDPIT or multidimensional gel protein identification system is an multidimensional liquid chromatography (LC)-based system of separation prior to tandem mass spectrometry (MS/MS). It is not necessary to purify proteins prior MuDPIT, although a reduction in protein complexity by some prior purification is helpful in obtaining interpretable spectra. Protein lysates are digested into peptides that are loaded onto a strong cation exchange (SCX) support. Peptides are sequentially eluted onto a reverse-phase column for a second separation. Eluted peptides from the reverse-phase column are electrosprayed into a tandem mass spectrometer for amino acid sequencing and identification of proteins in the sample.

---