One of the most widespread methods used in the biotech industry for purifying recombinant proteins is based on affinity chromatography. Here, the aim in both analysis and preparative work is to isolate a single protein in its purest form from the surrounding matrix of undesired proteins and other cell elements.
Biontex supplies a range of modified cross-linked agaroses which specifically bind to tagged proteins. The functioning of these fusion proteins is generally unimpaired by the tag. Our products utilize two methods of affinity chromatography:
This method is based on the specific binding of sixfold histidine-tagged fusion proteins (6×His) to iminodiacetic acid (IDA) of triple-chelated Ni2+ or Co2+ ions with covalent binding to agarose2.
The polyhistidine tag is generally introduced into the vector indirectly on the N- or C-terminal end of the protein by means of appropriate coding or PCR. After the fusion protein binds to the carrier, elution can be effected by reducing pH value on the one hand and by means of a metal complexing agent such as EDTA or Imidazol – which is similar to the amino acid histidine – on the other. Separation of the His tag can finally be achieved by means of exopeptidases; in most cases, activity is unimpaired by the polyhistidine tag.
Glutathion-S-transferases (GST) are a widely found class of enzymes which catalyze transfer of tripeptide glutathion (Glu-Cys-Gly) to endogenic or xenobiotic substrates. These glutathion-coupled substances are generally more water-soluble and can be excreted via the kidneys after further degradation. For this reason glutathion S-transferases play a vital role in metabolism and detoxification of toxins in organisms.
The tripeptide glutathion and its position in the active centre of glutathion S-transferase4
This method is based on the specific binding of glutathion S-transferase (GST)-tagged fusion proteins to glutathion covalently bound to agarose via the central thiol group. The GST sequence of 220 amino acids is introduced at the N-terminal end of the desired proteins via a corresponding DNA sequence of the encoding gene and expressed via a vector. After the fusion protein binds to the carrier, elution takes place with an excess of free glutathion which displaces the recombinant fusion protein from the agarose carrier. In addition, when integrating a thrombin bridge GST can be separated from the fusion protein by appropriate proteases (e.g. thrombin, Factor X). This can also be effected directly from the carrier without prior elution.
For protein purification, Biontex supplies GST (cat. no. R010), Ni-IDA (cat. no. R020) and Co-IDA agaroses (cat. no. R030) in various unit sizes. These agaroses have areas of application ranging from antigen production for in vitro diagnosis, antibody development or the production of vaccines or other adjuvants to applications in research laboratories. Ultra-simple protocols and maximum yields, directly usable for purification of proteins from raw bacterial lysate plus repeated regenerability are only some of the outstanding features of Biontex agaroses for protein purification.
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