Chromatographic Purification

Question: Describe about the Chromatographic Purification? Answer: Purification of Soluble His-Tagged Proteins The ClpA is the protein of our interest is an ATP dependend chaperon and regulatory component of ClpAP protein basically expressed in E.coli cells. This expression of recombinant protein could be analysed by presence of small size of amino acids i.e. 6 histidine tag which will be conjugated to its C or N terminus of protein. It can purify by using metal chelate affinity chromatography method having concept based on strongly binding affinity of Nickle (II)-nitriloteriacetic acid (Ni-NTA) and NTA act as chelating ligand. The 6 histidine ligand binds with Ni2+ ion to NTA through four ligand sites leaving two sites vacant for binding with 6 his- tag (Zhao et al, 2010). The strong affinity between Ni conjugate NTA with hexahistadine tag lowers the chances of unspecific binding of protein in Ni-NTA beads. The washing of column allows the highest affinity binding of hexahistadine tag protein with IMAC resin by nearly neutral condition buffer. The next step to elute the tagged protein, in th is step the His tag protein is eluted by elution buffer twice with different concentration. The elution should be done in concentration gradient where the first concentration should be low which remove the unspecific binding of protein and the second elution should be done with high concentration that is used to remove the hexahistadine tag protein from the Ni-NTA (Crowe et al, 1994). Finally, the prurity of isolated pure his-tagged protein will be identified using techniques like SDS-PAGE and 2-dimesion gel electrophoresis leading to identification of pure protein. References: Zhao X, Li G, Liang S (2013) Several Affinity Tags Commonly Used in Chromatographic Purification. J Anal methods Chem: doi: 10.1155/2013/581093 Crowe J, Dobeli H ,Gentz R, Hochuli E ,Stibber D, Henco K (1994) 6xffis-Ni-NTA Chromatography as a Superior Technique in Recombinant Protein Expression/Purification .Methods in Molecular Biology .Mol biotechnol 31:371-387