His-tagged Protein Expression

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.

NiCo21(DE3) Two-Step Purification

Purification workflow of target protein that has been expressed in the NiCo21(DE3) strain of E. coli.

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FAQs for His-tagged Protein Expression
Protocols for His-tagged Protein Expression
Improved Purity of His-tagged Proteins with NiCo21(DE3)
A) Expression of Glutamyl tRNA Synthetase (6-His) in NiCo21(DE3) Competent E. coli followed by Ni-NTA purification. Eluent (E) from a Ni-NTA column was passed over a chitin column. The protein of interest elutes in the flow through (FT), while the CBD-tagged metal binding proteins remain bound (B) to the chitin resin (NEB #S6651S). Purifications were performed according to manufacturers' recommended conditions. B) Contaminants ArnA, SlyD and Can are confirmed by Western blot using Anti-CBD Antibody (NEB #E8034S)
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