Scholarly article on topic ' Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification '

Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification Academic research paper on "Biological sciences"

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Academic research paper on topic " Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification "



Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification

Kasper R. Andersen, Nina C. Leksa, and Thomas U. Schwartz*

Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139


His-tag affinity purification is one of the most commonly used methods to purify recombinant proteins expressed in E. coli. One drawback of using the His-tag is the co-purification of contaminating histidine-rich E. coli proteins. We engineered a new E. coli expression strain, LOBSTR (low background strain), which eliminates the most abundant contaminants. LOBSTR is derived from the E. coli BL21(DE3) strain and carries genomically modified copies of arnA and slyD, whose protein products exhibit reduced affinities to Ni and Co resins, resulting in a much higher purity of the target protein. The use of LOBSTR enables the pursuit of challenging low-expressing protein targets by reducing background contamination with no additional purification steps, materials, or costs, and thus pushes the limits of standard His-tag purifications.

Proteins 2013; 81:1857-1861. © 2013 Wiley Periodicals, Inc.

Key words: BL21(DE3); E. coli protein expression strain; His-tag affinity purification; LOBSTR.


Many methods of recombinant protein purification have been developed. One of the most widely used techniques is the His-tag affinity purification.1 A small His-tag (usually 6 or 10 histidines) is fused to either the N or C terminus of the target protein, enabling capture by nickel or cobalt ions coordinated on a variety of commercially available resins. The small size of the His-tag, low cost, and ease of use have made it the most popular affinity-tag available. Expression of recombinant His-tagged proteins is largely carried out in Escherichia coli because it is easy to culture and it allows for the production of target proteins with high yield. However, one major drawback of His-tag affinity purification of proteins expressed in E. coli is the presence of naturally histine-rich host proteins, resulting in co-purification of these contaminants.2,3 The two most common E. coli

contaminants are ArnA, a bifunctional enzyme involved in the modification of lipid A phosphates with aminoarabi-nose,4 and SlyD, a peptidyl-prolyl ds/trans-isomerase.1,5 ArnA has several non-consecutive histidine residues, which are surface exposed and form clusters within the hexame-ric structure.2,3,6 In contrast, SlyD is characterized by a 48 amino acid unstructured C-terminal tail containing 15 histidines.4,7 Because the Ni-binding mechanism of ArnA

Additional Supporting Information may be found in the online version of this article.

Grant sponsor: Lundbeck Foundation and a Sapere Aude DFF postdoc grant (to K. R. A); Grant sponsor: NIH, Pew Scholar Award; grant number: GM077537 (to T.U.S.).

K. R. Andersen and N. C. Leksa contributed equally to this work. ^Correspondence to: Thomas U. Schwartz, Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. E-mail:

Received 26 March 2013; Revised 19 June 2013; Accepted 28 June 2013 Published online 15 July 2013 in Wiley Online Library ( DOI: 10.1002/prot.24364


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and SlyD mimics that of the His-tag, both proteins are co-purified along with the target protein in His-tag affinity purifications. For well-expressing recombinant proteins, these endogenous proteins are a small problem because they are out-competed by the sheer amount of the protein of interest. However, many proteins, including human proteins, large, multi-domain proteins, and co-expressed protein complexes are ignored as viable targets for in vitro studies because they express poorly and consequently cannot be isolated in sufficient amounts or with high purity. When protein expression is low, host proteins, especially ArnA and SlyD, have a similar abundance and compete for binding on Ni or Co resins. As a result, ArnA and SlyD are purified in nearly equal amounts when compared to the target protein. The most effective means to increase the purity of the target protein is to use additional affinity tags or multiple purification steps, however, this lowers the yield and increases the purification time and cost. Because both arnA and slyD knockout strains suffer growth defects, these strains are not viable options for recombinant protein expression.8,9 To address these problems, we designed a new E. coli expression strain named LOBSTR (low-background-strain), which features genomic modifications in arnA and slyD based on surface engineering. LOBSTR maintains normal cell growth but significantly reduces the Ni- and Co-binding affinities of both host proteins. LOBSTR drastically reduces ArnA and SlyD contamination, thus enabling the purification even of poorly expressing target proteins.


Wild-type arnA was PCR amplified from E. coli genomic DNA with Ndel and Xhol restriction site overhangs on the 5' and 3' ends, respectively, using primers 1F and 1R (See all primer details in Supporting Information Table S1), and cloned into the bacterial expression vector pColaDuet (EMD Millipore). Two serine point mutations were introduced at site 1 (H359S and H361S) using primers 2F and 2R. Two additional serine point mutations were introduced at site 2 (H592S and H593S) using primers 3F and 3R to generate the final arnA mutant containing a total of four histidine to serine mutations.

The arnA knockout strain was generated with the E. coli recombineering technique,10 using the pKD4 plas-mid as a template for the selectable marker and BL21(DE3) as the parental strain. The forward and reverse primers, 4F and 4R, were designed to maintain the reading frame of arnB, which shares its start codon with the stop codon of arnA within the arn operon11 (also called pmrHFIJKLM operon12). A slightly modified scheme was used to introduce the arnA mutant back into the arnA knockout strain at the original locus (Supporting Information Fig. S1). First, mutant arnA was amplified and combined with the amplified selectable marker in a second PCR step. The resulting PCR product

containing mutated arnA and the selectable marker was transformed into the arnA knockout strain for recombination using the k Red recombinase plasmid (pKD46). The selectable marker was eliminated using the FLP plasmid (pCP20). For the modification in slyD, the arnA mutant strain was transformed with a PCR product (generated using primers 5F and 5R) containing a selectable marker flanked by homologous overhangs that, after recombination, result in the elimination of the 46-residue C-terminal, histidine-rich segment of SlyD. Again, the selectable marker was later removed using pCP20. Proper genomic integration was confirmed by PCR and sequencing. The RIL plasmid (Agilent Technologies) encoding rare tRNAs was transformed into the final expression strain to improve the expression of our eukaryotic target proteins.

The binding affinity of wild-type and mutant ArnA were assessed by immobilizing purified protein onto a 1 ml His-Trap FF column (GE Healthcare) equilibrated in 50 mM potassium phosphate pH 8.0, 300 mM NaCl, and 5 mM beta-mercaptoethanol. Protein was eluted with a linear gradient of 0-150 mM imidazole. The imidazole concentration at the elution peak of each protein was recorded and compared.

Growth analysis was performed at 18, 25, and 37° C for both LOBSTR and the BL21(DE3) strains carrying the same test expression plasmid (Supporting Information Table S2 for a list of all test constructs). Cultures of 1 L were grown in LB medium supplemented with 0.4% (w/v) glucose and antibiotic selection at 37°C to 0D600 —0.7. Protein expression was induced with 0.2 mM IPTG 20 min after the cultures were shifted to the desired expression temperature. OD600 was measured from the initial synchronization time and until the cells were harvested —20-22 h after induction.

To test protein purification, BL21(DE3) and LOBSTR cultures were started at 37°C in LB medium supplemented with 0.4% (w/v) glucose and appropriate antibiotic selection. At 0D600 —0.7, cultures were shifted to 18° C and induced with 0.2 mM IPTG -20 min later. Cultures were harvested after 18-20 h. For each strain and construct tested, a total of —3.5 g of cells were resus-pended in 50 mL of resuspension buffer (40 mM potassium phosphate pH 8.0, 150 mM NaCl, 40 mM imidazole, and 3 mM beta-mercaptoethanol) and lysed with a cell disrupter (Constant Systems). Lysates were cleared for 25 min at 9500g and the soluble fraction was incubated with 400 iL bed volume of Ni Sepharose 6 Fast Flow (GE Healthcare) resin for 1 h while stirring at 4°C. The resin was collected and washed with 6 mL of resuspension buffer and eluted with 2 mL of elution buffer (40 mM potassium phosphate pH 8.0, 150 mM NaCl, 250 mM imidazole, and 3 mM beta-mercaptoethanol). Elution fractions were analyzed on a 4-15% SDS-PAGE gradient gel (Bio-RAD) and stained with Coomassie Blue R250. Purifications using Ni-NTA (Qiagen) and Talon

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(Clontech) resins were performed using resuspension buffer containing 20 mM or 5 mM imidazole, respectively, following manufacturer's recommendations.


We designed surface engineered forms of E. coli ArnA and SlyD based on their crystal and NMR structures, respectively6,7 (Supporting Information Fig. S2). Both proteins have exposed histidine-rich surfaces that result in binding to immobilized metal-affinity resins. ArnA is a hexamer, formed by a dimer of trimers. The structure revealed two prominent surface-exposed patches of histi-dine residues. One of the patches is at a trimer interface and results in a cluster of nine histidines per trimer (Supporting Information Fig. S2, site 1). We mutated histidine residues 359 and 361 to serines to abolish this histidine-rich surface. The second cluster of surface-exposed histi-dines was removed by mutating histidines 592 and 593 to serines (Supporting Information Fig. S2, Site 2). To determine whether the histidine to serine mutations resulted in weaker Ni-binding affinity, both recombinant wild-type and mutant ArnA were first purified in batch. Subsequently, pure protein was loaded onto a His-trap Ni-col-umn and eluted with a linear imidazole gradient. Wildtype ArnA eluted at a concentration of —60 mM imidaz-ole, while mutant ArnA showed significantly weaker binding affinity, eluting at —30 mM imidazole (Supporting Information Fig. S3). Thus, mutating four histidine residues to serines in ArnA (24 per hexamer) lowers the Ni-affinity to a level comparable to non-specific binding. Similarly, analysis of the SlyD NMR structure showed that all of the clustered histidine residues reside in an unstructured tail at the very C terminus of the protein (Supporting Information Fig. S2). A previous study suggested that deleting this tail has little effect on cell growth.13,14 Thus, we truncated SlyD at residue 150, thereby maintaining the structural integrity of the catalytic N-terminal domain while removing the entire unstructured tail. Using a modified recombineering10 approach, we then replaced the genomic copies of arnA and slyD in the host strain BL21(DE3) with our mutant versions to create LOBSTR (overview Supporting Information Fig. S1). To confirm that the combined genetic modifications in LOBSTR also maintain normal growth, we monitored and compared its growth rate to the parental BL21(DE3) strain at 18, 25, and 37°C. A test construct (See Supporting Information Table S2 for a list of all test constructs) was expressed over the duration of the growth analysis. No significant difference in growth rate at any of the induction temperatures was observed between LOBSTR and BL21(DE3), and the final 0D600 of the cultures after overnight induction were very similar (Fig. 1).

To verify that LOBSTR reduces ArnA and SlyD contamination, we performed small-scale purifications of seven different protein constructs in the parental

0 2 4 6 8 10 12 14 16 18 20 22

Time {Hours)

Figure 1

LOBSTR and the parental BL21(DE3) strain show comparable growth. The growth (OD600) of both LOBSTR and the parental BL21(DE3) strain was measured from initial synchronization at 0 h until the final harvest. Both strains carried the same expression plasmid and were grown at 37°C until an OD600 ~0.7, at which point protein expression was induced at 18, 25, and 37°C (black arrow). The growth curves for LOBSTR and BL21(DE3) are shown in red and black, respectively. Cell growth during log phase and final cell density was similar for both strains. Depending on the expression plasmid different growth behavior was observed, but typically no growth difference was seen between LOBSTR and BL21(DE3).

BL21(DE3) strain and in LOBSTR. The seven constructs (Supporting Information Table S2) were chosen to represent a wide range of potential targets, including low- and higher-expressing constructs, monomeric proteins, dimeric complexes, 6X- and 10XHis-tagged proteins. Most of our test constructs contain a SUMO-tag fused to the N terminus to increase protein solubility. In the BL21(DE3) strain background, high levels of contamination by both ArnA and SlyD can be seen in the elutions (Fig. 2). Illustrating the low expressions levels of target proteins, ArnA and SlyD are purified in amounts nearly equivalent to that of the target protein, as seen in constructs 2, 4, and 5. However, in LOBSTR, the vast majority of contaminants are eliminated, with the target protein now being the most prominent protein. Purification of construct 1, a heterodimeric complex with one binding partner carrying a 6X His-tag, is also greatly enhanced in LOBSTR. Furthermore, the amounts of all target proteins purified are similar between the BL21(DE3) strain and LOBSTR. Since the initial purity is much greater, fewer subsequent purification steps are required to obtain pure protein, resulting in equivalent, if not greater, final yields from LOBSTR. Curiously, a secondary contaminant, indicated by a double asterisk (**) in Figure 2, is also reduced

proteins 1859

27m~ — Ä«-SlyD

Figure 2

ArnA and SlyD are eliminated from His-tag purifications from LOBSTR. Elution samples of test purifications from BL21(DE3) and LOBSTR using common metal affinity resins are shown. (A) Seven protein constructs were purified from both the parental BL21(DE3) strain and LOBSTR using Ni Sepharose 6FF resin (GE Healthcare). The constructs are numbered 1-7, and contain either a 6XHis-tag (1 and 4) or a 10XHis-tag (2,3,5-7). See Supporting Information Table S2 for a list of all test constructs. The elution samples were run on an SDS-PAGE gel and stained with Coomas-sie Blue R250. ArnA and SlyD are indicated by arrows and target proteins indicated with a black circle (•). The double asterisk (**) indicates Hsp15, another protein showing reduced Ni-binding affinity in LOBSTR. (B) Purifications of constructs 1 and 5 from BL21(DE3) and LOBSTR were also carried out on two additional commonly used resins, Ni-NTA (Qiagen) and Talon (Clontech). In each case, ArnA and SlyD are successfully eliminated in LOBSTR.

in LOBSTR. This protein, identified by mass spectrometry as Hsp15, is reported to bind nucleic acids.15 While no modifications have been made to this protein in LOBSTR, we speculate that it may have non-specific binding affinity to SlyD, which is highly negatively charged. To ensure that the results seen here are reproducible on a variety of commercially available resins, we purified constructs 1 and 5 on two additional commonly used resins, Ni-NTA (Qia-gen) and Talon (Clontech) [Fig. 2(B)]. Both resin manufacturers recommend lower imidazole concentrations in the binding and washing buffers compared to the Ni Sepharose 6 FF resin (GE Healthcare), which was used for the purifications above. Still, nearly complete elimination of ArnA and SlyD contamination is observed on these resins as well [Fig. 2(B)].


LOBSTR enables the pursuit of poorly expressed protein targets in E. coli by lowering the background contamination of ArnA and SlyD. Previously, constructs yielding only 0.1-1 mg of target protein per liter of culture could be considered inadequate for in vitro studies. At such low levels of expression, ArnA and SlyD compete for the binding capacity of the metal affinity resin and are co-purified in equivalent or even greater amounts. LOBSTR enables a significantly higher yield and purity of poorly expressed target protein eluted from Ni or Co resins. Protein purity is of key importance for most downstream purposes, whether the protein is used in medical applications, binding studies, functional assays,

1860 proteins

or structural studies (EM, SAXS, NMR, and crystallography). An alternate approach to eliminate E. coli host contaminants has been developed previously.9 Here, ArnA, SlyD, and Can were genomically tagged with a chitin-binding domain and eliminated over chitin beads, pulling out the contaminants and leaving the target protein in the flow-through. In addition, GlmS is mutated to reduce binding to Ni and Co. While this method is successful in removing the contaminants, it requires an additional purification step as well as an additional resin, increasing both the time and cost of each purification. However, LOBSTR only requires a one-step purification to eliminate the major E. coli contaminants ArnA and SlyD with no additional costs and is specifically designed for low-expressing proteins. An alternate purification strategy is to simply perform a second IMAC step after cleaving off the His-tag from the protein of interest so that contaminants are rebound while the cleaved protein remains in the flow-through. While this method is successful when the contaminants make up only a small fraction of the total immobilized protein in the first IMAC step, it is highly inefficient if the contaminants are abundant and thus substantially reduce the initial yield. LOBSTR instead, incorporates genomic modifications to arnA and slyD in order to reduce the affinity of their gene products for metal affinity resins, eliminating them from co-purification with recombinant proteins of interest. Thus, proteins that were previously ignored as targets for recombinant expression and purification are now accessible.


We thank Anna Kaertner and Marta Carrara for initial help on the project. Simon Ringgaard for advice on recombineering and Greg Kabachinski for reading the manuscript.


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