Affiliation:
1. New England BioLabs, Inc., Gene Expression Division, 240 County Road, Ipswich, Massachusetts 01938
Abstract
ABSTRACT
Recombinant His-tagged proteins expressed in
Escherichia coli
and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native
E. coli
proteins, especially if the recombinant protein is expressed at a low level. The
E. coli
contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered
E. coli
BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two
E. coli
BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each
E. coli
CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The “NiCo” strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
97 articles.
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