Download Nickel affinity chromatography in Protein purification

Nickel affinity chromatography in
Protein purification
Date: 26/03/2015
Awaji-kansan Obediah
Contents
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Affinity Chromatography
Protein purification by Nickel affinity
Types of Ligands used
Advantages of some of the Ligands used
Applications
References
Affinity chromatography
Affinity chromatography has to do with the immobilisation
of a metal ion, usually a transition metal to a ligand which
is then used to separate or purify protein or other
biomolecules.
Nickel just like any other transition metal has affinity for
protein, specifically protein molecules containing Histidine
residue.
Affinity chromatography
 In order for affinity chromatography to work
perfectly the right choice of the ligand to be used is
very crucial,
 The metal ion should have a strong coordination with
the ligand as well as the protein of interest.
Affinity chromatography
 Points to note when choosing a ligand:
Stability of the ligand under the condition of
operation.
The interaction with the protein of interest should be
reversible. This is important because the protein has
to be separated from the ligand after purification
Affinity chromatography
Protein purification by Nickel
affinity
 Nickel affinity is specific for purification of protein
molecule containing histidine residue.
 One method used is IMAC (Immobilised-metal affinity
chromatography), with a centrally placed nickel ion.
Protein purification by Nickel affinity
Types of Ligands used
Nitrilotriacetic acid (NTA),
Iminodiacetic acid (IDA)
both could be used to purify proteins with histidine
molecules.
NTA coordinates the Ni2+ with four valences and two
valences are available for interaction with imidazole
rings of histidine.
Types of Ligands used
IDA coordinates Nickel with 3 valences while 3 other
electrons on Nickel are left to interact with the
protein.
Advantages/Disadvantages of some of
the ligands used
 high level of purity obtained with NTA
 This is due to the strong coordination between the
ligand and Ni2+ as well as the protein under
consideration.
 IDA has shown poor purity of protein this is due to
the fact that it has 3 coordination points with the
ligand and 2 with the protein
Advantages/Disadvantages of some
of the ligands used
 One of the electrons remains free and this leads to
leaching of Ni2+ which could bind to the surface of the
purified protein and lower the purity of the protein
after elution.
 A penta-dentate ligand can be used, this has lower
metal leaching but protein recovery is very low.
Applications
 Some of the applications of IMAC:
IMAC can be used to separate structurally intact and
soluble His-tagged antigens.
It can also be used to separate denatured protein
from the ones that are not denatured
References
 Jerker, P. and Bright, O. (1983). Immobilised metal affinity
adsorption and immobilised metal affinity chromatography of
biomolecules. 22(7), 1621-1630
 Sigma-Aldrich (). His-select Nickel affinity Gel. Retrieved from
https://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/Sigma/Bulletin/p6611bul.pdf
 http://www.iba-lifesciences.com/Double-tag_His-tag.html
 http://info.gbiosciences.com/blog/bid/200711/What-You-Need-toKnow-About-NTA-and-IDA-Ligands
Questions
 Thank you for your time