Metal affinity chromatography

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]. 

Suresh, V., Gallant, S., and Cramer, S., Immobilized metal affinity chromatography displacer characteristics of traditional mobile phase modifiers, Biotechnol. Prog., 12, 84, 1996. 

Patwardhan, A.V. and Ataai, M.M., Site accessibility and the pH dependence of the saturation capacity of a highly cross-linked matrix. Immobilized metal affinity chromatography of bovine serum albumin on Chelating Superose, /. Chromatogr. A, 767, 11, 1997. 

Wu, H. and Bruley, D.F, Homologous Human Blood Protein Separation Using Immobilized Metal Affinity Chromatography Protein C Separation from Prothrombin with Application to the Separation of Factor IX and Prothrombin, Biotechnol. Prog., 15, 928, 1999. 

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. 

Another means of moving beyond pure protein preparations to high-throughput characterization of proteomes is to enrich for phosphopeptides from complex mixtures by metal affinity chromatography (Andersson and Porath, 1986). Using this method, protein mixtures are proteolyzed to create peptides and phosphorylated peptides are enriched by metal affinity chromatography and subsequently identified by mass spectrometry. This method is limited, however, because in many cases phosphopeptides absorb poorly or nonphosphorylated peptides absorb nonspecifically to the metal affinity resins (Ahn and Resing, 2001). 

Lopatin, S.A., and Varlamov, V.P. (1995) New trends in immobilized metal affinity chromatography of proteins. Appl. Biochem. Microbiol. 31, 221-227. 

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.

Ficarro, S. B., Salomon, A. R., BriU, L. M., Mason, D. E., Stettler-GiU, M., Brock, A., and Peters, E. C., Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites, Rapid Communications in Mass Spectrometry 19(1), 57-71, 2005. 

Gaberc-Porecar V, Menart V. Perspectives of immobilized-metal affinity chromatography. Journal of Biochemical and Biophysical Methods 2001 49 335-360. 

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]. 

Phosphorylation on serine, threonine, and tyrosine residues is an extremely important modulator of protein function. Phosphorylation can be analyzed by mass spectrometry with enrichment of compounds of interest using immobilized metal affinity chromatography and chemical tagging techniques, detection of phosphopep-tides using mass mapping and precursor ion scans, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags (McLachlin and Chait 2001). 

Metal affinity chromatography is a relatively new method that separates proteins on the basis of metal binding. This technique is used in Experiment 4 to isolate a-lactalbumin from milk. 

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... 

A wide range of immobilization chemistries are commercially available in conjunction with Sepharose beads. We have investigated a limited subset of these possibilities which include direct, nonoriented immobilization via Schiff s base chemistry, oriented nonco-valent immobilization via immobilized metal affinity chromatography resins and oriented noncovalent immobilization via biotin-streptavidin binding. At present we favor direct, covalent attachment of proteins via primary amines since it is highly efficient (typically better than 85% yield), minimizes leaching and provides the best NMR results (Figure 6.2). 

Figueroa, A., Corradini, C., Feibush, B., and Karger, B., High-performance immobilized-metal affinity chromatography of proteins on iminodiacetic acid silica-based bonded phases, J. Chromatogr., 371, 335, 1986. 

Analytical Properties Separation of proteins by immobilized-metal affinity chromatography (HPIMAC) with Cu(ll) or Zn(ll) present in the mobile phase Reference 29... 

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. 

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