Choice of native or denaturing purification conditions and use of reducing agents with TALON resin
Native or denaturing?—Selecting the right purification conditions
Deciding whether to use native or denaturing purification conditions depends on protein localization, solubility, accessibility of the histidine tag, downstream applications, and whether preservation of biological activity is required. TALON resin retains its protein-binding specificity and yield under a variety of purification conditions. It is stable under both denaturing and native (nondenaturing) conditions.
Native conditions
Purifying a protein under native conditions (see example below) is the most efficient way to preserve its biological activity, but requires that the protein is soluble. Advantages include:
- Eliminating the renaturation step at the end of the purification, saving time, and preventing significant loss of activity
- Retaining the ability to copurify enzyme subunits, cofactors, and associated proteins
Native purification with TALON resin preserves the biological activity of proteins. Fresh cells (0.5 g) expressing 6xHis GFPuv were extracted in 5 ml of 50 mM sodium phosphate; 0.3 M NaCl, pH 7.0 Panel A. Elution profile of GFP which was loaded, washed with the same buffer, and then eluted with a step gradient of imidazole (150 mM). Panel B. Fractions were analyzed by SDS-PAGE. The fluorescent signal of green fluorescent protein (GFPuv) was completely enriched by TALON Superflow Metal Affinity Resin.
Denaturing conditions
Because proteins that are overexpressed in prokaryotic systems sometimes form insoluble aggregates called inclusion bodies, you may need to purify proteins under denaturing conditions (see example below)—using strong denaturants such as 6 M guanidinium or 8 M urea to enhance protein solubility. Advantages include:
- Complete solubilization of inclusion bodies and his-tagged proteins
- Improved binding to the matrix and reduced nonspecific binding, due to full exposure of the tag
Purification of 6xHis GFPuv under denaturing conditions using TALON resin. The fusion protein was purified in 8 M urea using TALON resin. M = molecular weight markers.
His-tagged proteins purified under denaturing conditions can be used directly in subsequent applications or may need to be renatured and refolded. Protein renaturation and refolding can be performed prior to elution from the column. However, yields of recombinant proteins will be lower than under native conditions, because urea and guanidinium molecules compete with histidines for binding to metal.
Use of reducing agents
Purification with TALON resin may be carried out in the presence of β-mercaptoethanol, but not DTT or DTE, to preserve reduced sulfhydryl (-SH) groups that are important for the biological activity and structure of a given protein.
Native purification of 6xHis protein using TALON resin in the presence of beta-mercaptoethanol. N-terminal 6xhis-tagged mouse DHFR (19.5 kDa) was expressed in E. coli. 2 ml of lysate was purified using gravity flow on TALON resin in increasing concentrations of beta-mercaptoethanol. Even lanes: 20 μl of nonadsorbed material. Odd lanes: 5 μl of eluate.
TALON provides higher yields than Ni-NTA in the presence of β-mercaptoethanol.
Protein purification yields in the presence of beta-mercaptoethanol with TALON resin compared to Ni-NTA resin. N-terminal 6xhis-tagged DHFR was expressed and purified under native conditions. Protein concentrations were determined by Bradford assay. Yields are expressed as a percentage of total protein in the cell lysate.
TALON resin application protocols
Denaturing purification
Choose between mini-scale purification, batch/gravity-flow column purification, and large-scale purification protocols.
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