His6 (aka His6) is used in synthetic techniques which makes use of a His6 tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a His6 tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis[1].
Chemical ligation enables the synthesis of large polypeptide chains by the chemoselective reaction of unprotected peptide segments. A variety of ligation chemistries has been developed for this purpose. Application of the chemical ligation principle has led to practical chemical syntheses of a wide variety of different classes of proteins. Synthetic access to protein molecules has been used to elucidate the molecular basis of protein folding and stability, to elucidate the molecular basis of protein function, to design and build proteins of novel structure, and to determine the molecular structure of proteins by both NMR and x-ray crystallography. Chemical protein synthesis has also been used to develop candidate protein therapeutic molecules with improved properties.
The native chemical ligation reaction is both practical and highly effective. Each ligation product can be purified by reverse-phase HPLC and characterized with great precision by electrospray MS. In principle, the ability to purify and characterize intermediate products at each successive stage of construction of a protein molecule is one of the major advantages of the chemical ligation approach to total protein synthesis; it ensures accurate construction of high-purity protein molecules. However, in practice, such purification and characterization are arduous: the consecutive chemical ligation of several peptide segments involves multiple laborious purifications carried out by reverse-phase HPLC (see Fig. 1A). The repetitive HPLC purifications result in significant handling losses. Moreover, each of these purification steps entails time-consuming lyophilization of the product to enable solvent exchange for subsequent reactions.
The HIS6 gene encodes N-(5'-phospho-D-ribosylformimino)-5-amino-1-(5''-phosphoribosyl)-4-imidazole carboxamide isomerase, which catalyzes the fourth step in histidine biosynthesis. The reactions and enzymes involved in histidine biosynthesis have been identified in many organisms, and are thoroughly reviewed in Alifano et al. Mutations in HIS6, as well as in genes encoding other histidine biosynthetic enzymes, cause histidine auxotrophy and sensitivity to copper, cobalt, and nickel salts.