Affinity tags summary
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Publicado: 2 de mayo de 2011
Affinity Tags
* There is a general perception that solubility problems can often be solved by using a eukaryotic host, such as insect cells (the baculovirus expression system) or yeast
*The most common method that is used to improve the solubility of recombinant proteins in E. coli is to reduce the temperature at which the target protein is being produced
*Solubility-enhancing affinity tags tend to be proteins rather than peptides
* The hexahistidine tag (His-tag), which binds to immobilized transition metals, is by far the most commonly used affinity tag forhigh-throughput protein purification.
* Processing efficiency can often be improved by using more protease over a longer period of time or by incorporating extra residues (e.g, polyalanine) adjacentto the protease recognition site.
Two Intein System
* Protein splicing is a post-translational process analogous to RNA splicing. In this process, inteins catalyze their selfsplicing from theprecursor protein with the concomitant ligation of flanking polypeptide sequences, termed exteins, via a native peptide bond. Intein-mediated protein purification is a convenient and cost-effectiveapproach, and it utilizes the inducible self-cleavage activity of an engineered intein to isolate the target protein
* The result of the SDS-PAGE indicated that C-terminal intein (intein2) fusionwas unstable. Intein2 underwent substantial in vivo cleavage, which produced CBD-Ssp-GFPmut3* and Mxe-CBD fragment
* The two-intein is capable of isolating proteins with an amino terminalresidue other than methionine and free of a fusion tag
Recombinogenic Engineering
* Two alternative approaches, RecA-dependent engineering and ET recombination, can be used to insert, delete orsubstitute DNA sequences at any desired position on a target molecule
* Homologous recombination allows the exchange of genetic information between two DNA molecules
* Because homologous...
* There is a general perception that solubility problems can often be solved by using a eukaryotic host, such as insect cells (the baculovirus expression system) or yeast
*The most common method that is used to improve the solubility of recombinant proteins in E. coli is to reduce the temperature at which the target protein is being produced
*Solubility-enhancing affinity tags tend to be proteins rather than peptides
* The hexahistidine tag (His-tag), which binds to immobilized transition metals, is by far the most commonly used affinity tag forhigh-throughput protein purification.
* Processing efficiency can often be improved by using more protease over a longer period of time or by incorporating extra residues (e.g, polyalanine) adjacentto the protease recognition site.
Two Intein System
* Protein splicing is a post-translational process analogous to RNA splicing. In this process, inteins catalyze their selfsplicing from theprecursor protein with the concomitant ligation of flanking polypeptide sequences, termed exteins, via a native peptide bond. Intein-mediated protein purification is a convenient and cost-effectiveapproach, and it utilizes the inducible self-cleavage activity of an engineered intein to isolate the target protein
* The result of the SDS-PAGE indicated that C-terminal intein (intein2) fusionwas unstable. Intein2 underwent substantial in vivo cleavage, which produced CBD-Ssp-GFPmut3* and Mxe-CBD fragment
* The two-intein is capable of isolating proteins with an amino terminalresidue other than methionine and free of a fusion tag
Recombinogenic Engineering
* Two alternative approaches, RecA-dependent engineering and ET recombination, can be used to insert, delete orsubstitute DNA sequences at any desired position on a target molecule
* Homologous recombination allows the exchange of genetic information between two DNA molecules
* Because homologous...
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