Protein affinity chromatography selectivity

A variety of chromatography techniques is available for separation and analysis of proteins, i.e., RP, lEX, size exclusion, hydrophobic interaction, and affinity chromatography. Selection of the type of chromatography, scale of operation, and applied detection technique will be based on the sample availability and complexity and, on the other hand, the required information. 

Affinity chromatography using factor XII as ligand leads to purification of u-PAR rather selectively, with only trace quantities of cytokeratin 1 or gClqR present [K. Joseph and A. Kaplan, unpubl. observations]. It is of interest that none of these three proteins possesses a transmembrane domain but u-PAR has a phos-phatidylinositol linkage within the cell membrane. Nevertheless, each of them has been isolated from purified cell membranes and they have been demonstrated to exist within the cell membrane by immunoelectron microscopy [41] presumably... 

Affinity chromatography exploits the high selectivity of most proteins for their ligands. Enzymes may be puri-... 

Before the era of artificially recombinant DNA, affinity chromatography emerged as a potentially highly selective approach to protein purification [13]. It revived a laborious art based on selective precipitations and a limited range of chromatographic media. 

Affinity chromatography and related techniques (e.g., thiol chromatography and IMAC) are widely used for preparative isolation because they enable a single protein or class of proteins to be selectively purified from very complex mixtures. They may be occasionally used as analytical tools. For example, protein A affinity chromatography has been used for quantitative analysis of immunoglobulins in ascites fluid.45 Information about surface-accessible histidine and phosphate groups may be obtained using IMAC. 

The first step in biopharmaceutical development is the selection of a clone in a specific cell line. Whole-mass analysis, if possible, is a fairly simple and powerful tool at this stage to verify the successful expression and translation of the desired protein. VanAdrichem et al.65 described the use of MALDI MS to monitor protein expression in several mammalian cell lines like CHO DXB11, CHO SSF3, and hybridomas. Quantitative MALDI-TOF MS measurements of an IgG antibody and insulin during large-scale production in hybridoma cells were comparable to affinity chromatography results. 

In affinity chromatography, the resin contains especially selected molecules that will interact with the particular polymer(s) that is being studied. Thus, for a particular protein, the resin may be modified to contain a molecule that interacts with that protein type. The solution containing the mixture is passed through the column and the modified resin preferentially associates with the desired protein, allowing it to be preferentially removed from the solution. Later, the protein is washed through the column by addition of a salt solution and collected for further evaluation. 

Table 3.19. Some forms of affinity chromatography that may be employed to purify a selected protein. Virtually all proteins carry out their biological effects by interacting in some way with other molecules, which can be given the general title ligands . This interaction is often quite biospecific. Immobilization of such ligands (or molecules which mimic ligands) onto chromatographic beads thus facilitates selective purification of the molecule of interest...

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