Affinity-column purification

Figure 9.21 Isotope Coded Affinity Tagging as an approach for comparative proteomics. Two pools of peptides can be differentially labelled with tags of different masses, then the pools combined, enriched by avidin affinity column purification (avidin has a very high affinity for biotin) and relative abundance of particular peptides can be directly compared by mass spectrometry (Reproduced from Patterson and Aebersold, 2003, Fig. 5).

Compounds 13 and 20 were obtained in five and seven steps respectively from the intermediate alcohol 6. They, as well as the acetylated derivative 12, were tested in vivo i and proved to be, as their free acids, gratuitous inducers of pectinases in Erwinia chiysanthemi. These results provide evidence that the hydroxyl function in C-5 is not required for the recognition between inducers and the KdgR repressor protein. Tharefore we can now envisage the preparation of affinity columns, by immoWlizing inducers on suppc rts using a spacer arm boimd to the C-5 hydroxyl. Such columns could then be used for the purification of the repressor protein. 

SIAB and sulfo-SIAB have been used to make a high-capacity RNA affinity column for the purification of human IRP1 and IRP2 (Allerson et al., 2003), to couple antibodies or Fab fragments to amine-modified microparticles (Harma et al., 2000), and in the attachment of oligonucleotides to surfaces for detection arrays (Adessi et al., 2000). 

Figure 16.5 A catch-and-release ICAT design incorporates a gem-methyl group and an isopropyl group on either side of a disulfide bond within its spacer arm. The hindered disulfide permits the use of standard reducing gel electrophoresis conditions using DTT without reduction. After purification on a (strept)avidin affinity column, however, the disulfide group can be cleaved with TCEP, which provides recovery of the labeled peptides prior to mass spec separation.

The following protocol for EPL, including purification using a CBD fusion tag followed by native chemical ligation, is based on the methods of Muir et al. (1998), Chong et al. (1997, 1998), Evans et al. (1998), Severinov and Muir (1998), and the NEB instruction manual for the IMPACT-TWIN system. The recombinant protein is recovered from the affinity column as the thioester derivative ready for reaction with a N-terminal Cys peptide or another tag containing a Cys residue. 

Baues, R.J., and Cray, G.R. (1977) Lectin purification on affinity columns containing reductively ami-nated disaccharides./. Biol. Chem. 252, 57. 

Chong, S., Mersha, F.B., Comb, D.G., Scott, M.E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H., and Xu, M.-Q. (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192, 271-281. 

Alternatively, a terminal tag such as polyhistidine can be added to the target protein to improve the efficiency of the purification procedure [24]. An affinity column designed to recognize polyhistidine could be used to isolate the tagged protein.The polyhistidine is then cleaved and the mixture dialyzed. While this approach is useful for small-scale preparations, whether it can be used for large pharmaceutical scale preparations remains to be seen. 

Sun, L., Ghosh, I., Xu, M.Q. (2003). Generation of an affinity column for antibody purification by intern-mediated protein ligation. J. Immunol. Methods, 282(1-2), 45-52. 

Fry, M.J. Panayotou, G. Dhand, R. Ruiz-Larrea, F. Gout, I. Nguyen, O. Courtneidge, S.A. Waterfield, M.D. Purification and characterization of a phosphatidylinositol 3-kinase complex from bovine brain by using phos-phopeptide affinity columns. Biochem. J., 288, 383-393 (1992)... 

The purification of the pancreatic alpha amylase was effected on an affinity adsorbent prepared from enzymically degraded starch plus agarose activated with bisoxirane.14 The fractions from the affinity column were analyzed for protein components by u.v. absorbance, and for alpha amylase activity by incubating the fractions with starch and measuring the increase in reducing sugars. The results are shown in Fig. 5. 

---