A New Tandem-Affinity Tag for Two-Step Protein Purification under Fully Denaturing Conditions

Tech ID: 18833 / UC Case 2005-046-0

Background

Preservation of posttranslational modifications during purification is crucial for successful mass spectrometric analyses of protein modifications. Current tandem-affinity purification strategies require native conditions and are therefore susceptible to loss of posttranslational modifications during cell lysis and purification because modifying as well as de-modifying enzymes remain active under these conditions.

Technology Description

University of California, Irvine researchers have developed a novel tandem-affinity tag that is compatible with two-step purification under fully denaturing conditions such as 8 M urea or 6 M guanidinium. This novel tag serves as a biotinylation signal in vivo. Tagged proteins are efficiently biotinylated in vivo in both yeast and mammalian cells at a specific lysine residue present in the tag. The biotinylated protein can then be sequentially purified by Ni2+-chelate chromatography and binding to streptavidin resins.

Applications

Taken together, this novel tag and its derivatives are useful tools for proteomic studies using mass spectrometry. The tag allows tandem affinity purification under fully denaturing conditions and thus combines a high degree of purification with preservation of protein modifications in the in vivo status. In addition, the procedure is compatible with in vivo cross-linking, which allows identification of transiently associated proteins, and can thus be an important tool to probe and understand the dynamics of the proteome.

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