His-Tag for Purification of Recombinant Proteins: A hexa-His sequence is called a His-Tag. It has been shown that an amino acid sequence consisting of 6 or more His residues in a row will also act as a metal binding site for a recombinant protein. The 6xHis affinity tag facilitates binding to immobilized Ni. 6xHis coding sequence can be placed at the C- or N-terminus of the protein of interest and recombinant protein with 6xHis residues can be expressed. The tag is poorly immunogenic, and at pH 8.0 the tag is small, uncharged, and therefore does not generally affect secretion, compartmentalization, or folding of the fusion protein within the cell. In most cases, the 6xHis tag does not interfere with the structure or function of the purified protein as demonstrated for a wide variety of proteins, including enzymes, transcription factors, and vaccines. Sometime, a protease cleavage site is inserted between protein sequence and His-tag. After His-tag affinity purification, the purified protein is treated with the specific protease to remove His-tag. A very common example is His -tagged Proteins with Thrombin, a protease, cleavage site.

Thrombin recognizes the consensus sequence Leu-Val-Pro-Arg-Gly-Ser, cleaving the peptide bond between Arg and Gly. This is utilised in many vector systems which encode such a protease cleavage site allowing removal of an upstream domain (Fig. 2)