Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography

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.

Fig. 1.

Purification of alanyl-tRNA synthetase (AlaRS), expressed in BL21(DE3) and BL21(DE3) slyD-CBD, by Ni²⁺ affinity chromatography. (A) Coomassie blue-stained SDS-PAGE gel of elution fractions obtained from a cell extract of BL21(DE3) expressing His-tagged AlaRS. (B) Coomassie blue-stained SDS-PAGE gel of elution fractions obtained from a cell extract of BL21(DE3) slyD-CBD expressing His-tagged AlaRS. (C) Western blot analysis of fractions shown in panel B probed with anti-CBD antibody. Lane M, molecular mass marker in kDa; lane FT, flowthrough after Ni-NTA; lanes 1 to 23, eluted fractions from the IMAC column using a gradient from 25 mM to 250 mM imidazole. AlaRS, wild-type SlyD (SlyDwt), and SlyD-CBD are indicated by a dash.

Fig. 2.

Purification of alanyl-tRNA synthetase (AlaRS), expressed in BL21(DE3) and BL21(DE3) slyD-CBD, on Ni²⁺ affinity chromatography followed by chitin bead treatment. The left panel represents SDS-PAGE analysis, and the right panel shows a Western blot analysis performed with anti-CBD antibodies. Lane M, molecular mass marker in kDa; lane L, cell lysate load; lane P, pool of Ni-NTA fractions 4 to 24; lane FT_C, flowthrough of chitin beads; lane B, boiled chitin beads. AlaRS, wild-type SlyD (SlyDwt), and SlyD-CBD are indicated by arrows.

Fig. 3.

BL21(DE3) glmS-CBD and GlmS mutant characterization. (A) Representation of the GlmS protein showing the two enzymatic domains, the two active sites, and the locations of the histidines (His) selected for replacement by alanines. Numbers indicate the amino acid residues. (B) Bacterial growth assay of BL21(DE3) and BL21(DE3) glmS-CBD and complementation assays performed with BL21(DE3) glmS-CBD carrying pMAK705 (empty vector), pMAKglmS, or pMAKglmS6Ala. The assay was carried out by spotting 5 μl of each overnight culture, diluted from 10⁻² to 10⁻⁷, on LT medium with or without N-acetylglucosamine (GlcNAc; 200 μg/liter). The plates were then incubated 24 h at 30°C. (C) SDS-PAGE of elution fractions obtained after loading cell lysates containing GlmS or mutant GlmS onto a 1 ml HisTrap HP column. This affinity test of GlmS, GlmS_2Ala, and GlmS_6Ala for Ni-NTA was performed using cell lysate from 200 ml culture of each strain grown at 30°C and induced with 0.5 mM IPTG for 17 h at 20°C. Lane M, molecular mass marker in kDa; lane −, BL21(DE3) expressing GlmS from the chromosome (empty plasmid); lane wt, BL21(DE3) overexpressing GlmS from pMAK705; lane 6Ala, BL21(DE3) expressing GlmS_6Ala from pMAK705; lane 2, BL21(DE3) overexpressing GlmS_2Ala from pMAK705. The position of GlmS is indicated by an arrow. The table indicates the number of spectra of GlmS identified by mass spectrometry analysis in each elution fraction. The presented ratio was estimated after normalizing the total protein spectral intensity value obtained for GlmS to that for the control sample “−.”

Fig. 4.

Growth curves and detection of CBD-tagged proteins. (A) Western blot analysis (anti-CBD) of total cell extracts from three independent clones (1, 2, 3) of NiCo21(DE3) and NiCo22(DE3). The CBD-tagged proteins are indicated by arrows. (B) Growth of BL21(DE3), NiCo21(DE3), and NiCo22(DE3) performed at 30°C in LB medium.

Fig. 5.

Activity assays for SlyD-CBD, ArnA-CBD, and AceE-CBD. (A) SlyD-CBD activity is confirmed by lysis sensitivity of BL21(DE3), NiCo21(DE3), and NiCo22(DE3) upon induction of protein E. Cultures of these strains harboring the plasmid pMS119-pE or pMS119 were induced at an OD₆₀₀ of 0.15 to 0.55 with 500 μM IPTG, indicated by an arrow, and monitored for optical density. (B) Bacterial growth assays with BL21(DE3), NiCo21(DE3), and NiCo22(DE3) were carried out by spotting 5 μl of overnight culture, diluted from 10⁻² to 10⁻⁷, on LB medium with or without 2 μg/ml polymyxin B. The plates were incubated for 24 h at 30°C or 37°C. (C) Bacterial growth assays with BL21(DE3), NiCo21(DE3), NiCo22(DE3), and two aceE mutants, CGSC5477 and CGSC4823, were carried out by spotting 5 μl of overnight culture, diluted 10⁻² to 10⁻⁷, on minimal medium with 0.2% glucose and supplemented with 2 mM potassium acetate when indicated.

Fig. 6.

Purification of the glutamyl-tRNA synthetase (GluRS) expressed in NiCo21(DE3) and NiCo22(DE3). Strains BL21(DE3), NiCo21(DE3), and NiCo22(DE3) expressing GluRS(6His) from pET21a were propagated in a high-density fermentor as indicated in Materials and Methods. One gram of each cell pellet was processed to purify GluRS(6His) using 1 ml Ni-NTA superflow resin followed by 1 h of batch incubation on chitin beads (1 ml). (A) Samples collected during the procedure were analyzed by SDS-PAGE. Lane M, molecular mass marker in kDa; lane L, cell lysate load; lane FT, flowthrough; lane W, first wash; lane B, boiled chitin beads; lane E, Ni-NTA elution. (B) Western blot analysis (anti-CBD) of the samples shown in panel A. GluRS, wild-type SlyD (SlyDwt), SlyD-CBD, ArnA-CBD, Can-CBD, and AceE-CBD are indicated by a symbol or an arrow. (C) The table indicates the number of spectra of each protein identified by mass spectrometry analysis in the elution fractions after Ni-NTA and in the flowthrough after chitin bead treatment.

Fig. 7.

Utility of the NiCo strains for poorly expressed proteins. Coomassie blue-stained SDS-PAGE gel of samples taken during the purification of GluRS on Ni-NTA superflow resin (2 ml) followed by 30 min of batch incubation on chitin beads (2 ml). The purifications were performed from a mixed cell lysate containing 1 ml GluRS lysate and 15 ml of empty-vector lysate from the same strain. Lanes 1, 4, and 8 represent lysate mixtures loaded (L) onto Ni-NTA columns. Each mixture contains a low concentration of GluRS which becomes the prominent band in the 250 mM imidazole elution sample (E). ft, Ni-NTA flowthrough; FT_C, sample taken of the void volume after incubating the Ni-NTA elution sample with chitin beads.