PMID:21602383
| Citation |
Robichon, C, Luo, J, Causey, TB, Benner, JS and Samuelson, JC (2011) Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography. Appl. Environ. Microbiol. 77:4634-46 |
|---|---|
| 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. |
| Links |
PubMed PMC3127686 Online version:10.1128/AEM.00119-11 |
| Keywords |
Biotechnology/methods; Chromatography, Affinity/methods; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/isolation & purification; Escherichia coli Proteins/metabolism; Genetic Engineering; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism |
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