Bio-affinity chromatography

While there are no set protocols for the specific order in which the different chromatographic separations are carried out, ion exchange and hydrophobic interaction chromatography typically precede gel filtration and bio-affinity chromatography. The latter can involve expensive resins and is often performed after various contaminants have been removed during earlier purification steps. 

Fig. 9.1. Schematic illustration of the elements involved in bio-affinity chromatography. The solute (Lt) is retained on the stationary phase (S) by specific interaction with the ligand (Ln). The ligand is covalently attached to a spacer arm (SP) which is in turn attached to the stationary phase. Elution as shown here is achieved by specific ligand competition by another solute (Lt ). Alternatively the ligand-ligate complex can be disrupted by reversible denaturation using low pH solvent or mild chaotropic salts, e.g.

Sepharose (e.g. Sepharose CL and Bio-Gel A) is a bead form of agarose gel which is useful for the fractionation of high molecular weight substances, for molecular weight determinations of large molecules (molecular weight > 5000), and for the immobilisation of enzymes, antibodies, hormones and receptors usually for affinity chromatography applications. 

Avidin (from egg white) [1405-69-2] Mr -70,000. Purified by chromatography of an ammonium acetate soln on CM-cellulose [Green Biochem J 101 774 1966]. Also purified by affinity chromatography on 2-iminobiotin-6-aminohexyl-Sepharose 4B [Orr 7 Bio/C/iew 256 761 1981]. It is a biotin binding protein. 

Affinity chromatography, which is the binding of bio-molecules with the matrix bed, often used for antibodies and antigens... 

Figure 6.2 A Likely purification procedure for tPA produced in recombinant E. coLi cells. The heterologous product accumulates intracellularly in the form of inclusion bodies. In this particular procedure, an ultrafiltra-tion step is introduced on several occasions to concentrate the product stream, particularly prior to application to chromatographic columns. Lysine affinity chromatography (Lys-chromatography) is employed, as tPA is known to bind immobilized Lysine molecules. Adapted with permission from Bio/Technology (1993), 11, 351... 

Clonis, Y. D. (1990). Large scale affinity chromatography. Bio/Technology 5, 1290. 

Table 1 Comparison of the recombinant production and affinity chromatographic purification of GFP from B. megaterium [30]. Different affinity tag fusion forms of GFP were produced in II. megaterium WH323. Purification was performed using affinity chromatography. Amounts of purified GFP-Strep were determined using a Bradford protein assay kit (Bio-Rad Munich Germany) and BSA (Perbio Rockford USA) as standard. Amounts of purified GFP-His, His-TEV-GFP, Strep-Xa-GFP and Strep-TEV-GFP were calculated via their relative fluorescence per mg protein...

Affinity separation of proteins in immobilized metal ion affinity chromatography (IMAC). The method is based on the fact that some protein molecules, having specific residues on the surface, can form complexes with metal ions immobilized on the sorbent matrix. Retention is determined by the coordination interactions forming the complexes. Immobilized metal ion affinity chromatography can be performed under very mild, nondenaturing conditions with extremely high selectivity it is particularly suitable for preparative group fractionation of complex extracts and bio-fluids. 

IgG in antiserum prepared as described above is purified by affinity chromatography. To prepare the affinity column, conjugate the antigen (component I or II) to Affi-Gel 15 (Bio-Rad Laboratories) according to the manufacturer s Instruction Manual. 

Affinity Chromatography is performed on a unique stationary phase which has a specific bio-active hgand bonded onto a solid support. It is mostly used with bio-molecules and only the active component of a sample is attracted to the stationary phase (e.g., wheat germ lectin for polysaccharides and soybean trypsin inhibitor for proteases). The remaining chemicals are washed off and... 

Table 16-1. Affi-Gel supports for affinity chromatography. (Courtesy - Bio-Rad Laboratories, Richmond, CA)...

One challenge in process chromatography applied to biologically active or derived molecules is that these molecules exist with many other co-products, particularly if the desired product molecule is fermentation derived (5). In a separation scheme, a target molecule can be separated from other components based on differences in shape or size, ionic character, hydrophobic character, and/or bio-affinity (6). The resin s capacity to interact with the molecule is a key parameter, and is described by the capacity factor ... 

Affinity chromatography separation effected by affinity of solute molecules for a bio-specific stationary phase consisting of complex organic molecules bonded to an inert support material, e.g. separation of proteins on a bonded antibody stationary phase. The technique is really selective filtration rather than chromatography. 

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