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His-tagged Protein Expression
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Application Overview
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.
Featured Products
NiCo21(DE3) Competent E. coli
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FAQs for His-tagged Protein Expression
FAQs
Protocols
Improved Purity of Histagged Proteins with
NiCo21(DE3)
Legal Information
What systems does NEB offer for protein expression and purification?
What are the advantages of magnetic affinity matrices?
What are the strain properties of NiCO21(DE) competent E. Coli?
Protocols for His-tagged Protein Expression
Protein Expression with T7 Strains
Protocol for Removal of IMAC Contaminating Proteins (C2529)
Legal and Disclaimers
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New
England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require
the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at
[email protected].
This product is intended for research purposes only. This product is not intended to be used for therapeutic or
diagnostic purposes in humans or animals.
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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