Metal affinity purification

Figure 5 Effect of varying the multiplicity of infection (MOI) on the overall levels of expression and the efficiency of crosslinking between a pair of single-cysteine mutants of ACKR3 and CXCL12. (A) Nonreducing 10% SDS-PAGE of the samples following metal affinity purification by the tag on the receptor. The lower and the upper bands indicate non-crosslinked receptor and crosslinked complex, respectively. (B) Band densitometry for quantification of receptor expression levels. Receptor yield is expressed as fold increase over the control sample (receptor only, no chemokine). Although the calculated fraction of crosslinked complex is close to 80% across all samples, the receptor yield is maximal at the MOI of 6 4 (receptorrchemokine).

Following metal affinity purification, promising candidate complexes are analyzed for yield and crossUnking efficiency by nonreducing SDS-PAGE and Western blotting. These assays appear central in crosslink characterization as they provide the proper context for interpretation of all other experiments. 

If protein degradation is a problem, include a protease inhibitor cocktail (exclude EDTA from cocktail when using metal affinity purification because this will strip NE+ or Co from the column). Also, for some proteins, it may be desirable to include dithiothreitol (DTT) in the purification, wash, and elution buffers. This is not routinely done for plant poly (A) factor purification as DTT inhibits the activity of the Arabidopsis CPSF30 protein [18]. 

Stiborova, H., Kostal, J., Mulchandani, A., Chen, W. (2003). One-step metal-affinity purification of histidine-tagged proteins by temperatnre-triggered precipitation. Biotechnology and Bioengineering, 82, 605-611. 

The ELP expression system was compared to the conventional oligohistidme fusion, which is traditionally applied for purification by immobilized metal affinity chromatography (IMAC). Both techniques were shown to have a similar yield of the recombinant protein. The temperature-triggered approach offers a fast and inexpensive nonchromatographic separation with the possibility for larger scale purification. Although the ELP expression system may not be applicable to all types of recombinant proteins, numerous examples have already been shown [40]. 

The endothelin B receptor is an example of characterization of a homogeneous, affinity purified protein (Roos et al., 1998). Significant progress has been made in the development of techniques for more high-throughput identification of phosphorlyation events. Analysis of large sets of phosphorylated proteins is facilitated by the availability of affinity purification methods such as anti-phosphotyrosine or anti-phosphoserine antibodies or metal affinity chromatography (Neubauer and Mann, 1999 Soskic et al., 1999). These methods are not specific to a particular protein but rather are used to fractionate all proteins that are phosphorylated. 

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). 

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.

Among these amino acids histidine is the most commonly used one. Attachment of histidine tags to the recombinant proteins polypeptides is the most known development in the field of IMAC. Histidine and other metal affinity tags are widely used for protein purification [26], Adsorbents may be prepared by binding chelators onto the surface and metals to the chelators. Free coordination sites of the metal ions are needed for the analyte to bind to metal ions [25]. 

Ji Z, Pinon DI, Miller LJ. Development of magnetic beads for rapid and efficient metal-chelate affinity purifications. Analytical Biochemistry 1996 240 197-201. 

The inhibition of SuSy by the divalent cations (fCi 15 aM), Zn (fC 25 aM), and Ni (fti 37 aM) was exploited for further purification of SuSyl by immobilized metal affinity chromatography (IMAC). A subsequent gel filtration yielded homogeneous SuSyl suitable for crystallization experiments [24]. The protein chemical characterization revealed a homotetrameric organization of the 93 kDa subunit. Our kinetic data for the cleavage reaction and preliminary immunoblot analysis for phosphoserine suggested that SuSyl may be phosphorylated in the yeast expression system [28]. 

Metal affinity columns can be used for the purification of antibodies with a hexa-Histidine tag. Immobilized metal affinity chromatography is incompatible, in our own experience, with direct loading of antibodies in supernatants... 

CYE Wu, LC Blaszczak, MC Smith, PL Skatrud. Construction of a modified penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus and purification by immobilized metal affinity chromatography. J Bacteriol 176 1539-1541, 1994. 

Kumar A, Bansal V, Andersson J, Roychoudhury PK, Mattiasson B (2006), Super-macroporous cryogel matrix for integrated protein isolation - immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line, J. Chromatogr. A 1103 35-42. 

Hale, J. E., and Beidler, D. E. (1994). Purification of humanized murine and murine monoclonal antibodies using immobilized metal-affinity chromatography. Anal. Biochem. 222, 29-33. 

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