Protein overexpression and affinity purification are used in various biotechnology research fields and play an important role in the post genomic era. While many of the common purification techniques make use of tags like 6XHis and GST, they face drawbacks such as significant background and an extra tag-removal step (especially if the tag is known to interfere with the protein’s function).
Bio-Rad’s Profinity eXact protein purification system utilizes an immobilized engineered protease that specifically recognizes and binds the Profinity eXact tag with high affinity. Following purification, the protein is washed and the tag is cleaved while still on-column.
This technical note demonstrates how the Profinity eXact purification system overcomes concerns common to expression and purification systems in eukaryotic cells including: poor tag expression, tag degradation, posttranslational modification and misfolding of tag and excessive background contamination. The results indicate that the Profinity eXact system can be used in eukaryotic cells for single-step purification of tag-free proteins with low background and without compromising the obvious function of the system.