Affinity purification of protein

Walker PA, Leong LE, Ng PW, Tan SH, Waller S, Murphy D, Porter AG. 1994. Efficient and rapid affinity purification of proteins using recombinant fusion proteases. Biotechnology 12 601-605. 

Strategies for the De Novo Design of Biomimetic Ligands for the Affinity Purification of Proteins... 

Cheeseman, I.M. and Desai, A. (2005) A combined approach for the localization and tandem affinity purification of protein complexes from metazoans. Sci. STKE 2005, 11. 

Fig. 3. Intein-mediated protein ligation. The IMPACT system allows affinity purification of proteins fused to an intein-CBD tag and their further isolation with a C-terminal thioester moiety (A), or an N-terminal cysteine (B). (A), N-terminal intein splicing for thioester isolation. Target protein (protein 1) is expressed in E. coli with C-terminally located intein-CBD tag. After specific binding to the chitin resin, the thiol reagent provokes the cleavage of the peptide bond between the target protein and the intein. Whereas the intein-CBD tag remains bound to the chitin resin, the protein thioester is eluted from the column. (B), C-terminal cleavage to obtain N-terminally...

Figure 7 Affinity purification of protein based on Ni-His-tag interaction.

Recently we attempted to use our monosize PS particles as a carrier matrix for affinity purification of proteins. We selected albumin as a potential model protein. We studied both nonspecific albumin adsorption on the PS particles and also specific albumin adsorption on the dye-attached PS particles (49,61). Some interesting results of these studies are briefly discussed below. 

After affinity purification of proteins using the functionalized beads, bound proteins need to be digested for further LC/MS analysis. Two different strategies are presented and their advantages are briefly discussed. 

We have employed a more systematic random mutagenesis approach by dividing the BDI into consecutive clusters of 10 amino acid residues (Boxes) that are individually replaced with a string of 10 alanine residues in the full-length Hisg-tagged allele to facilitate affinity purification of the mutant protein (Valasek et ah, 2004). Alternatively, clusters rich in... 

Bergseid, M., Baytan, A.R., Wiley, J.P., Andener, W.M., Stolowitz, M.L., Hughes, K.A., and Chesnut, J.D. (2000) Small molecule-based chemical affinity system for the purification of proteins. BioTechniques 29, 1126-1133. 

Metal chelate affinity chromatography finds most prominent application in the affinity purification of recombinant proteins to which a histidine tag has been attached (described later). As protein binding occurs via the histidine residues, this technique is no more inherently useful for the purification of metalloproteins than for the purification of non-metalloproteins (a common misconception, given its name). 

After affinity purification, the protein usually has high purity however some non-specific binding to the resin can occur, along with binding of any truncations of the target protein. 

Zou El, Tuo Q, Zhou D. Affinity membrane chromatography for the analysis and purification of proteins. Journal of Biochemical and Biophysical Methods 2001 49 199-240. 

Affinity chromatography is widely used as a means of separation and purification with specific properties. It represents one of the most effective methods for the purification of proteins as well as many other molecules. For example, Loog et al. 

The availability of a great variety of group-specific adsorbents in prepacked columns makes possible the combination of FPLC and affinity chromatography for the separation and purification of proteins. 

Much less commonly used than partition, ion exchange, and size columns, affinity columns are of growing interest in the HPLC purification of proteins... 

Recently, active recombinant a-LTX has been generated using bacteria in which both thioredoxin reductase and glutathione reductase are inactivated to improve the formation of disulphide bonds in expressed proteins (Li et al. 2005). The toxin is expressed as a fusion with glutathione-S-transferase (GST), which is used for affinity purification of the recombinant toxin and can be subsequently removed by selective proteolysis. Considering the relative ease of generating recombinant proteins in bacteria, this approach will facilitate structure-function studies of a-LTX. 

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