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Potential for Using Histidine Tags in Purification of Proteins at Large Scale
Author(s) -
GabercPorekar V.,
Menart V.
Publication year - 2005
Publication title -
chemical engineering and technology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.403
H-Index - 81
eISSN - 1521-4125
pISSN - 0930-7516
DOI - 10.1002/ceat.200500167
Subject(s) - nitrilotriacetic acid , histidine , chemistry , iminodiacetic acid , chelation , combinatorial chemistry , hexa , chromatography , metal ions in aqueous solution , cleavage (geology) , metal , enzyme , organic chemistry , biochemistry , materials science , fracture (geology) , composite material
Attachment of oligo‐histidine tag (His‐tag) to the protein N‐ or C‐terminus is a good example of early and successful protein engineering to design a unique and generalized purification scheme for virtually any protein. Thus relatively strong and specific binding of His‐tagged protein is achieved on an Immobilized Metal‐Ion Affinity Chromatography (IMAC) matrix. Most popular hexa‐histidine tag and recently also deca‐histidine tag are used in combination with three chelating molecules: iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), and carboxymethyl aspartic acid (CM‐Asp), covalently attached to the chromatographic matrix. The following combinations with divalent metal ions are preferentially used: (Cu, Zn, Ni, Co)‐IDA, Ni‐NTA, and Co‐CM‐Asp. At large scale, regarding cost and product purity, a decisive step is precise and efficient cleavage of His‐tag by the cleavage enzyme. Two‐step IMAC followed by a polishing step appears to be a minimum but still realistic as an approach to generic technology also for more demanding products. Possible drawbacks in using His‐tags and IMAC, such as leaching of metal ions, inefficient cleavage, and batch‐to‐batch reproducibility must be carefully evaluated before transferred to large scale. Although a great majority of reports refer to small laboratory scale isolations for research purposes it appears there is much higher potential for more extensive use of His‐tags and IMAC at large scale than currently documented.