Compare and contrast the Ni-NT

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

Name three general techniques or tools used in the Ni-NTAchromatography

What is histidine? Why is His6 such a popular proteintag for purification?

Summarize how the scientists produced the different variants ofHis6-GFP protein samples for purification.

How do we use His6/Ni-NTA interactions inexperiments?


1. The Ni-NTAPurification System is an affinity based purification system solelydesigned for purification of 6xHis-tagged recombinant proteinsexpressed in bacteria, insect, and mammalian cells whereas HIC is aform of chromatographic technique which separates protein moleculesusing the properties of hydrophobicity.

The system is designed around thehigh affinity and selectivity of Ni-NTA Agarose for recombinantfusion proteins that are tagged with six tandem histidine residueswith a complete system that includes purification buffers and resinfor purifying proteins under native, denaturing, or hybridconditions. On the other hand hydrophobic interactionchromatography (HIC) is one of the most widely used methods forseparating and purifying proteins in their native state. HIC alsoproves to be quite useful in isolating protein complexes and instudying protein folding and unfolding.

The resin used in Ni-NTA exhibitshigh affinity and selectivity for 6xHis-tagged recombinant fusionproteins and in HIC high affinity is exhibited towards hydrophobicproteins.

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

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

Denaturingconditions are used if the protein is insoluble (in thepellet after lysis) or if the downstream application does notdepend on protein activity.

The hybrid protocolis used if the protein is insoluble and the protein activity needsto be preserved.

3.Histidine, usually encoded by codonsCAU and CAC, is an ?-amino acidthat is used in the biosynthesis of proteins. It contains an?-amino group with protonated –NH3+ formunder biological conditions, a carboxylic acid group withdeprotonated –COO? form under biological conditions, andan imidazole side chain which remians partially protonated. It isclassified as a positively charged amino acid at physiologicalpH.

His6 tag, also known as polyhistidine tag is most commonly used in the production ofrecombinant proteins since the string of histidine residues bindsto several types of immobilized ions (such as nickel, cobalt andcopper) under specific buffer conditions to allow for the simpledetection and purification of His-tagged proteins. Polyhistidinetag is now considered as the most widely used affinity tag for avariety of protein purification purposes because of its relativelysmall size, low immunogenicity, hydrophilic nature and versatilityin the presence of detergents and many other additives, and undernative and denaturing conditions. In addition to this, its has theability to detect and purify recombinant proteins without using aprotein-specific antibody or probe, and the availability ofanti-His tag antibodies for use in assays involving His-taggedproteins make 6xHis tags all the more popular.

4. unable toanwer

5. The 6xHis (polyhistidine)/Ni-NTA system has become a fast and versatile tool forthe affinity purification of recombinant proteins and antigenicpeptides. It is based on the high-affinity binding ofsix-consecutive histidine residues (6xHis tag) which remainimmobilized nickel ions ultimately giving rise to a highlyselective interaction that allows purification of tagged proteinsor protein complexes from about 1% to >95% homogeneity. Thetight association between the tag and the resin alse leads to easywashing of the contaminants that too under stringent conditions,yet the bound proteins can be gently eluted by competition withimidazole, or a slight reduction in pH. 6xHis labeled proteins canbe purified even under the strongly denaturing conditions becausethe interaction is independent of the tertiary structure of thetag. These are required to solubilize inclusion bodies The sixhistidine residues that comprise the 6xHis tag can be attached ateither end of the recombinant protein, are uncharged atphysiological pH, and are very poorly immunogenic in all speciesexcept monkeys. This is how it is used in experiments. It iscurrently used in a wide variety of applications, ranging from thelarge-scale purification of proteins for antibody production, tothe isolation of subunits and substrates through their interactionswith the tagged proteins.

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