Question

Compare and contrast the Ni-NTA chromatography experiment and the HIC technique .

Name three general techniques or tools used in the Ni-NTA chromatography

What is histidine? Why is His₆ such a popular protein tag for purification?

Summarize how the scientists produced the different variants of His₆-GFP protein samples for purification.

How do we use His₆/Ni-NTA interactions in experiments?

Answer 1

1. The Ni-NTA Purification System is an affinity based purification system solely designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and mammalian cells whereas HIC is a form of chromatographic technique which separates protein molecules using the properties of hydrophobicity.

The system is designed around the high affinity and selectivity of Ni-NTA Agarose for recombinant fusion proteins that are tagged with six tandem histidine residues with a complete system that includes purification buffers and resin for purifying proteins under native, denaturing, or hybrid conditions. On the other hand hydrophobic interaction chromatography (HIC) is one of the most widely used methods for separating and purifying proteins in their native state. HIC also proves to be quite useful in isolating protein complexes and in studying protein folding and unfolding.

The resin used in Ni-NTA exhibits high affinity and selectivity for 6xHis-tagged recombinant fusion proteins and in HIC high affinity is exhibited towards hydrophobic proteins.

2. There are three different techniques to purify 6xHis-tagged proteins :

Native conditions are used if the protein is soluble (in the supernatant after lysis) and if the protein activity needs to be preserved.

Denaturing conditions are used if the protein is insoluble (in the pellet after lysis) or if the downstream application does not depend on protein activity.

The hybrid protocol is used if the protein is insoluble and the protein activity needs to be preserved.

3. Histidine, usually encoded by codons CAU and CAC, is an ?-amino acid that is used in the biosynthesis of proteins. It contains an ?-amino group with protonated –NH₃⁺ form under biological conditions, a carboxylic acid group with deprotonated –COO^? form under biological conditions, and an imidazole side chain which remians partially protonated. It is classified as a positively charged amino acid at physiological pH.

His6 tag, also known as poly histidine tag is most commonly used in the production of recombinant proteins since the string of histidine residues binds to several types of immobilized ions (such as nickel, cobalt and copper) under specific buffer conditions to allow for the simple detection and purification of His-tagged proteins. Polyhistidine tag is now considered as the most widely used affinity tag for a variety of protein purification purposes because of its relatively small size, low immunogenicity, hydrophilic nature and versatility in the presence of detergents and many other additives, and under native and denaturing conditions. In addition to this, its has the ability to detect and purify recombinant proteins without using a protein-specific antibody or probe, and the availability of anti-His tag antibodies for use in assays involving His-tagged proteins make 6xHis tags all the more popular.

4. unable to anwer

5. The 6xHis (poly histidine)/Ni-NTA system has become a fast and versatile tool for the affinity purification of recombinant proteins and antigenic peptides. It is based on the high-affinity binding of six-consecutive histidine residues (6xHis tag) which remain immobilized nickel ions ultimately giving rise to a highly selective interaction that allows purification of tagged proteins or protein complexes from about 1% to >95% homogeneity. The tight association between the tag and the resin alse leads to easy washing of the contaminants that too under stringent conditions, yet the bound proteins can be gently eluted by competition with imidazole, or a slight reduction in pH. 6xHis labeled proteins can be purified even under the strongly denaturing conditions because the interaction is independent of the tertiary structure of the tag. These are required to solubilize inclusion bodies The six histidine residues that comprise the 6xHis tag can be attached at either end of the recombinant protein, are uncharged at physiological pH, and are very poorly immunogenic in all species except monkeys. This is how it is used in experiments. It is currently used in a wide variety of applications, ranging from the large-scale purification of proteins for antibody production, to the isolation of subunits and substrates through their interactions with the tagged proteins.