Poly-histidine tagging is widely employed to aid in the purification of recombinant target protein. Corresponding nickel and cobalt containing resins offer a convenient means to isolate the His-tagged target protein. However, this method suffers from the fact that microbial expression hosts naturally express significant levels of divalent metal binding proteins. Furthermore, many of the endogenous metal binding proteins are essential for cell viability so they cannot be eliminated by a simple gene deletion approach.
The NiCo21(DE3) protein production strain was designed and engineered to improve the outcome of His-tagged protein purification. Three endogenous proteins (SlyD, ArnA and carbonic anhydrase (Can)) were tagged at the C-terminal end with the chitin binding domain (CBD). CBD tagging enables rapid removal of these contaminant proteins by exposing the cell lysate or target protein pool to chitin beads either before or after metal chelate chromatography. A fourth contaminant protein was mutated to eliminate binding to nickel or cobalt resin; six surface histidine of GlmS were mutated to alanine without affecting the function of the protein. The net result is that the NiCo21(DE3) strain exhibits the same growth characteristics and protein production potential as BL21(DE3) albeit with distinct advantages for recovering pure target protein structure-function studies.
FAQs for His-tagged Protein Expression
Protocols for His-tagged Protein Expression
Competent Cells Brochure
The Competent Cell brochure provides information on the different competent cell strains for cloning and protein expression available from NEB.
- Bypassing Common Obstacles in Protein Expression
- Competent Cell Product Comparison
- Convenient Formats of Competent Cells
Other Tools & Resources
Improved Purity of His-tagged Proteins with NiCo21(DE3)
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