Nickel affinity chromatography

Nickel affinity chromatography was chosen as the primary purification technique because it is a fast and reliable one-step assay and purified complexes can often be used in downstream applications without the necessity of removing the polyhistidine tag. In addition, the polyhistidine tag is smaller than many other affinity tags targeted by commercially available affinity resins and, in most cases, does not seem to interfere with the structure and function of the recombinant protein. 

Purification of humanized monoclonal antibodies has also been published using nickel affinity chromatography.190 Directly after filtration of feed stock, antibodies were adsorbed and elution was achieved by a descending pH gradient from 7.5 to 4.25. Purity obtained was about 90% and the fraction did not contain free albumin nor free light chains. Recovery was also reported as greater than 9Q% from ascitic fluids as well as from cell culture supernatants. 

The His6-SMT3-chemokine can be isolated from E. coli proteins and cell debris through nickel affinity chromatography. 

Chromatography matrices. DEAE-cellulose (DE-52, Whatman) hydroxylapatite (Bio-gel HTP, Bio-Rad), Mono Q anion exchange columns (Pharmacia Fine Chemicals) and Ni-NTA nickel affinity chromatography support (Qiagen)... 

Fusion rCRALBP produced with the pET 19b vector contains 23 additional amino-terminal residues, including 10 adjacent histidine residues (Fig. 1) that facilitate protein punfication by nickel-affinity chromatography. Purify with Qiagen Ni-NTA resin according to the supplier s instructions... 

One limitation to this method should be noted. If the antibody-enzyme conjugate is prepared using antibody fragments such as Fab or F(ab )2, then nickel-chelate affinity chromatography will not work, since the requisite Fc portion of the antibody necessary for complexing with the metal is not present. 

Figure 20.15 An affinity chromatography support containing iminodiacetic acid groups chelated with nickel may be used to remove excess enzyme after reactions to produce antibody-enzyme conjugates. The nickel chelate binds to the antibody in the Fc region, retaining the conjugate while allowing free enzyme to pass through the gel unretarded.

Porath, J., and Olin, B. (1983) Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Serum protein affinities for gel-immobilized iron and nickel ions. Biochemistry 22, 1621-1630. 

Figure 3 Biosynthesis and purification of 90-kD elastin analogue analyzed by denaturing polyacrylamide gel electrophoresis (10-15% gradient, visualized by silver staining). Lanes 1-7 time course of target protein expression at 0, 30, 60, 90, 120, 150, and 180 minutes after induction. Lane 9 soluble lysate of induced E. coli expression strain BLR(DE3)pRAMl. Lanes 10-13 protein fractions obtained from immobilized metal affinity chromatography of the lysate on nickel-NTA agarose (imidazole gradient elution). Lanes 8,14 protein molecular weight standards of 50, 75, 100, and 150 kD.

Most biocatalytic conversions are performed with the enzyme immobilized in the microreactor. Miyazaki et al. [426] developed a simple noncovalent immobilization method for His-tagged enzymes on a microchannel surface. These enzymes contain a polyhistidine-tag motif that consists of at least six histidine residues, often located at the N- or C-terminus. The H is-tag has a strong affinity for nickel and can be reversibly immobilized by a nickel-nitrilotriacetic acid (Ni-NTA) complex (Scheme 4.103), a strategy commonly used in affinity chromatography. 

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