Screening Kit for the purification of AMP binding proteins
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A characteristic of many proteins is their ability to bind specific small molecules (ligands) non-covalently with high affinity. This protein-ligand interaction can be used for rapid purification of a protein by affinity chromatography. In this technique, a ligand (e.g. a nucleotide) is immobilized onto the surface of a matrix (e.g. Agarose), which is incubated with a protein mixture to be purified. The protein of interest will bind to its ligand whereas other contaminants will not. These contaminants can be washed off, and the protein of interest can be eluted by an excess of free ligand in the elution buffer.
A ligand commonly used for this technique is AMP (Adenosine-5’-monophosphate) that is suitable (when immobilized onto an agarose matrix) for the purification of various NADH/NADPH- dependent dehydrogenases, ATP-dependent and AMP-binding enzymes as well as HIT family proteins (Tab. 1).
There is however, a fundamental problem with using AMP in affinity chromatography: For attachment to a matrix AMP needs to be chemically modified with a linker (Fig. 1). This linker may interfere with the protein-AMP interaction and thereby reduce the binding.
This problem can usually be circumvented by attaching AMP at a different position at the adenine base, the sugar or the phosphate moiety. Each of these linkage strategies has a characteristic effect upon protein-AMP interactions (Fig. 1).
The AMP Affinity Test Kit contains a set of 4 typical AMP-Agarose chromatography materials as well as the unmodified (blank) Agarose as a negative control.
6AH-AMP-Agarose and 8AHA-AMP-Agarose contain AMP that is immobilized via the adenine base but varies by the actual position of the linker (C6 and C8, respectively). αAH-AMP-Agarose and EDA-AMP-Agarose are phosphate and sugar modified derivates, respectively (Fig. 2).
With these four materials, the ideal material for purification of a particular protein of interest can be identified in a simple screening experiment.
Properties of AMP-Agaroses
|Bead size||45 - 165 μm|
|Degree of substitution||5 μmol/ml Agarose for αAH-AMP-, 6AH-AMP-, 8AHA AMP- and EDA-AMP-Agarose|
|pH stability (short term)||4 - 9|
|pH stability (long term)||7.5|
|Chemical stability||Stable to all solutions commonly used in gel filtration including 8 M urea and 6 M guanidine hydrochloride.|
General experimental remarks
The optimal purification procedure varies depending on the protein of interest.
Williams (1999) provides general background information on the usage of AMP agaroses that may be used as a starting point for the set up and optimization of your individual purification procedure.
The following proteins have been successfully purified with AMP Agaroses:
Table 1: AMP-Agaroses purified proteins
|Aminoaldehyde dehydrogenase||Sebela (2000)|
|Glutamate dehydrogenase||Brodelius (1997)|
|NADH Cytochrome b5 dehydrogenase||Manabe (1996)|
|Lactate Dehydrogenase||Pettit (1981)|
|Isocitrate dehydrogenase||Nealon (1979)|
|S-Nitrosoglutathione (GSNO) Reductase||Liu (2001 )|
|ATP-dependent & AMP-binding enzymes||Reference|
|aminoacyl-tRNA synthetases||Fromant (1981)|
|Glycogen phosphorylase||Sorensen (1975)|
|HIT protein family||Reference|
Bretes et al. (2013) Hint2, the mitochondrial nucleoside 5'-phosphoramidate hydrolase, properties of the homogeneous protein from sheep (Ovis aries) liver. Acta Biochim. Pol. 60 (2):249.
Manzer et al. (2003) Molecular cloning and baculovirus expression of the rabbit corneal aldehyde dehydrogenase (ALDH1A1) cDNA. DNA Cell Biol. 22 (5):329.
Liu et al. (2001) A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans. Nature 410 (6827):490.
Sebela et al. (2000) Characterisation of a homogeneous plant aminoaldehyde dehydrogenase. Biochim. Biophys. Acta 1480 (1-2):329.
Williams (1999) Miscellaneous Biospecific Affinity Gels. Protein Liquid Chromatography Chapter 20:731.
Manabe et al. (1996) Two novel mutations in the reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase gene of a patient with generalized type, hereditary methemoglobinemia. Blood 88 (8):3208.
Borgese et al. (1987) Concentration of NADH-cytochrome b5 reductase in erythrocytes of normal and methemoglobinemic individuals measured with a quantitative radioimmunoblotting assay. J. Clin. Invest. 80 (5):1296.
Falke et al. (1981) Adenylic acid: deoxythymidine 5'-phosphotransferase: evidence for the existence of a novel herpes simplex virus-induced enzyme. J. Gen. Virol. 53 (2):247.
Fromant et al. (1981) Affinity chromatography of aminoacyl-tRNA syntheses on agarose-hexyl-adenosine-5'-phosphate. Biochimie 63 (6):541.
Pettit et al. (1981) Purification of lactate dehydrogenase isoenzyme-5 from human liver. Clin. Chem. 27 (1):88.
Brodelius et al. (1979) Studies of bovine liver glutamate dehydrogenase by analytical affinity chromatography on immobilized AMP analogs. Arch. Biochem. Biophys. 194 (2):449.
Nealon et al. (1979) Purification and subunit structure of nicotinamide adenine dinucleotide specific isocitrate dehydrogenase from Neurospora crassa. Biochemistry 18 (16):3616.
Sorensen et al. (1975) Purification of glycogen phosphorylase by affinity chromatography on 5'-AMP Sepharose. Biochem. Biophys. Res. Commun. 67 (3):883.