BcMag™ Low Expression His-tagged Protein Purification Kit

A polyhistidin tag, also called 6xHis-tag, His-tagged, and His-tag, is a versatile tool for purifying the highly purified recombinant protein from various expression systems, including bacterial, yeast, plant cell, and mammalian cells systems. The tag comprises six or more consecutive histidine amino acid residues positioned at either N or C terminus of a recombinant protein. Due to its small size, His-tag has several distinctive features, including less immunogenicity, hydrophilic nature, and versatility under native and denaturing conditions. Additionally, it is unnecessary to cleave the tag from the recombinant protein since it rarely perturbs the structure and function of its fusion protein. The purification principle of the His-tag depends on immobilized metal ion affinity chromatography (IMAC). Immobilized metal ion affinity chromatography (IMAC) is a rapid affinity purification chromatography where the his-tagged protein is separated based on their affinity for Ni 2+ or Co 2+ that have been immobilized by a chelator to a solid matrix such as beaded agarose or column. At pH 7-8, his tagged protein will bind to Ni 2+ or Co 2+ . The binding reaction with the tagged protein is affected by various independent variables such as pH, temperature, salt type, salt concentration, immobilized metal and ligand density, and protein size. The bound protein is eluted by a decreasing pH gradient, increasing imidazole concentration, or adding an EDTA chelator in a buffer. This technique is an ideal tool for capturing and purification of his-tagged recombinant protein in a quick, inexpensive, and straightforward manner. Nonspecific binding was a potential problem with the purification of his-tagged proteins from various expression systems, especially from the lower expressed recombinant his-tagged proteins or higher expressed proteins interacting with other cellular proteins. Many factors can cause nonspecific binding. Among them, low expressed protein or the tagged protein bound with the endogenous proteins could result in severely nonspecific binding. The binding of His-tagged proteins to the metal ion of the IMAC depends on electric charges. The nonspecific binding occurs when the his-tagged binding sites of resin are only partially bound by the protein of interest due to its low abundance. The rest of the binding sites were non-specifically interacted with other slightly charged proteins such as histidine-rich protein, leading to impurities later on. Multiple lines of evidence prove that reducing reagents and nonionic detergents in the binding and washing buffers could dramatically reduce the highly nonspecific binding and get purer proteins. The most used chelators in IMAC applications are iminodiacetate (IDA) or nitrilotriacetic acid (NTA). These chelators cannot use together with reducing reagents such as DTT (dithiothreitol), β-me ( β-Mercaptoethanol), and TCEP (TRIS (2-carboxyethyl) phosphine) since the reducing reagents can strip off the metal from these resins, resulting in the rapid loss of the protein binding capacity. Moreover, those chelators are mainly immobilized to the traditional affinity chromatography matrices such as agarose resin or column. These solid matrices make the purification process tedious, time-consuming, unable to handle very tiny samples, and challenging to adapt to the automation system. Bioclone introduces a powerful magnetic bead based IMAC system to address these problems.

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