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

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

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.

TALON provides higher yields than Ni-NTA in the presence of β-mercaptoethanol.