Protein his-tagged

The second way of using complexes is to use a crystallization scaffold (Derewenda, 2004). Some proteins will not crystallize on their own, but will form crystals as part of a complex. This complex does not have to be of any biological relevance, except to promote crystallization. The first way of doing this is to make use of the affinity tag used to purify a protein (if one was used). His-tagged proteins are very commonly used for purifying proteins, and could be left on for crystallization studies. This is not usually a useful scaffold, as the poly-His plus linker sequence is usually very flexible. 

In protein microarrays, capture molecules need to be immobilized in a functional state on a solid support. In principle, the format of the assay system does not limit the choice of appropriate surface chemistry. The same immobilization procedure can be applied for both planar and bead-based systems. Proteins can be immobilized on various surfaces (Fig. 1) (12). Two-dimensional polystyrene, polylysine, aminosilane, or aldehyde, epoxy- or thiol group-coated surfaces can be used to immobilize proteins via noncovalent or covalent attachment (13,14). Three-dimensional supports like nitrocellulose or hydrogel-coated surfaces enable the immobilization of the proteins in a network structure. Larger quantities of proteins can be immobilized and kept in a functional state. Affinity binding reagents such as protein A, G, and L can be used to immobilize antibodies (15), streptavidin is used for biotinylated proteins (16), chelate for His-tagged proteins (17, 18), anti-GST antibodies for GST fusion proteins (19), and oligonucleotides for cDNA or mRNA-protein hybrids (20). 

His-tagged GUS-fusion proteins have been produced and isolated from tobacco chloroplasts. His-tagged proteins have also been extracted by foam frachonahon (Crofcheck et al., 2003,2004) or by a modihed intein expression system (Morassutti et al., 2002). 

Avoid introducing air bubbles. Slowly pour slurry down a thin glass rod inserted into empty column. The column and bed sizes depend on the amount of His-tagged protein to be purified. Generally, the binding capacity of Ni-NTA superflow is 5-10 mg protein per mL resin Ni-NTA superflow is supplied as 50% slurry. 

Usually, 5-10 column volumes are sufficient. Monitor pressure at this step. If the lysate is very viscous, the pressure may exceed the recommended value. Reduce the flow rate accordingly. Start with a flow rate of 0.5—1 mL/min if the His-tagged protein does not bind, the flow rate should be reduced. The flow rate may, however, be increased for protein elution. Collect fractions for SDS-PAGE analysis. 

If desired, a step gradient of elution buffer in wash buffer may be used to elute the protein. Five column volumes at each step are usually sufficient. The His-Tagged protein usually elutes in the second and third column volume. Imidazole absorbs... 

Lindner P, Bauer K, Krebber A, Nieba L, Kremmer E, Krebber C, Honegger A, Klinger B, Mocikat R, Pluckthun A, Specific detection of his-tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions, Bio techniques, 22 140-149, 1997. 

Purification of His-tagged protein under denaturing conditions... 

The same procedure is used for binding of His-tagged proteins to Ni-NTA, except the binding buffer must not contain DTT and EDTA. DTT may be replaced with 10 mM j8-mercaptoethanol. 

SlyD of E. coli combines an amino-terminal FKBP domain with a carboxyl-terminal His-rich domain, which binds metal ions. It was discovered because it is involved in cell lysis after phage infection (Maratea et al., 1985 Roof et al., 1997), but also because it binds tightly to immobilized Ni2+ and thus copurifies with His-tagged proteins (Wulfing et al., 1994). The prolyl isomerase activity of SlyD is regulated by Ni2+ binding to the His-rich domain (Hottenrott et al., 1997). 

Metal chelate affinity chromatography is a kind of separation method which has, as a ligand, a metal ion. Some proteins and peptides are purified on the basis of affinity for metal ions immobilized by chelation on the adsorbents. Histidine and cysteine form complexes with the chelated metals around neutral pH. Biological proteins include many histidines as well as recombinant proteins as polyhistidine fusions for instance, His-tag proteins have a specific metal chelate affinity. The adsorbent is prepared by coupling a metal chelate ligand with an iminodiacetic acid group, which forms a chelate with divalent metal ions such as Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+, Fe2+, etc. 

His-tag proteins produced by a recombinant are easily purified by metal chelating chromatography. His-tag proteins have about 6 histidines at the N- or C-terminal site and the His-tag easily forms a chelate with Ni2+, Zn2+ and Cu2+ Elution of His-tag proteins is carried out by increasing the concentration of imidazole in the buffer solution. 

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