Tagged proteins, purification

Wu WY, Fong BA, Gilles AG, Wood DW (2009) Recombinant protein purification by selfcleaving elastin-like polypeptide fusion tag. Curr Protoc Prot Sci 26.4 21-18... 

Due to the importance of this technology for protein expression, manufacturers Sigma, Serotec, Abeam and many others offer a wide range of products for the detection, isolation and purification of tagged proteins. These vendors also provide monoclonal and polyclonal antibodies specific for the most commonly... 

Protocol 1.3 Purification of soluble Hisg-tagged protein on Ni-NTA agarose... 

Leave to express for 3-4 days, then collect medium, spin, filter 0.22 pm, then go on to protein purification (method depends on the tags used etc.). 

Figure 2.3 Schematic representation of the workflow for automated protein purification strategies. ( offline tag cleavage involves a manual step.)...

Sigrell, J. A., Eklund, R, Gatin, M., Hedkvist, L., Liljedahl, P, Jotiansson, C. M., Pless, T. and Torstenson, K. (20(B). Automated multi-dimensional purification of tagged proteins. J. Struct. Funct. Genomics 4,109-114. 

Janknecht, R., G.de Martynoff, J.Lou, R. A.Hipskind, A.Norandeim and H.G.Stunnenberg. (1991) Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia vims. Proc Natl Acad Sci USA, 88, 8972-8976. 

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]. 

Alternatively, a terminal tag such as polyhistidine can be added to the target protein to improve the efficiency of the purification procedure [24]. An affinity column designed to recognize polyhistidine could be used to isolate the tagged protein.The polyhistidine is then cleaved and the mixture dialyzed. While this approach is useful for small-scale preparations, whether it can be used for large pharmaceutical scale preparations remains to be seen. 

High-Throughput Purification of Hexahistidine-Tagged Proteins Expressed in E. coli... 

Purification of the proteins from the cultures grown in 24-well blocks can be performed in 96-well filter plates using a vacuum manifold for column forming, washing, and elution steps, as outlined below. The hexahistidine-tagged proteins in cleared cell lysates are batch loaded onto Ni-NTA agarose in a 96-well filter plate. Columns are formed by applying low (200 mbar) vacuum pressure. These mini-columns are extensively washed, and subsequently the proteins are eluted into a microtiter plate. 

Detailed structure-function analysis of a-LTX is impossible without generating mutants. The difficulty inherent to this approach is to ensure proper folding of this large protein. Two groups relied on baculovirus expression and successfully purified active recombinant toxins. Ichtchenko et al. (1998) used two 8-histidine tags for purification, whereas Volynski et al. (1999, 2003) utilized a monoclonal antibody. 

In cases where it has proved impractical to assay for activity at each step, protein purification has been aided by tagging a fraction of the molecules with a photoaffinity reagent (e.g. the lactose carrier of Escherichia coli Newman et al., 1981 and the /J-adrenergic receptor of frog erythrocytes Shorr et al., 1982). 

Scheich C, Sievert V, Biissow K (2003), An automated method for high-throughput protein purification applied to a comparison of His-tag and GST-tag affinity chromatography, BMC Biotechnol. 3 12-19. 

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