Articles
Cobalt-based Protein Purification Resin Does Not Bind E. coli SlyD, A Common Contaminant in Ni-NTA IMAC
Mon, 02/09/2024 - 7:21am
by Jonathan L. McMurry and Robert M. Macnab
E. coli SlyD, a prolyl isomerase, specifically binds divalent metal ions, which can result in significant contamination of IMAC preparations of heterologously expressed His-tagged proteins. Purification of His-tagged proteins by immobilized metal affinity chromatography (IMAC) is a widely used method for producing large quantities of highly pure protein in a one-step process. The two most commonly used metal ions, Co2+ and Ni2+, are contained within BD TALON™ Resin and an Ni-NTA resin, respectively. Both can be used to purify His-tagged proteins under native and denaturing conditions.
SlyD, an E. coli prolyl cis/trans-isomerase, binds and is regulated by divalent metal ions.(1) It contains a C-terminal histidine-rich metal binding domain (Figure 1). SlyD possesses a high affinity for nickel ions,(3) presenting a potential problem for Ni-based IMAC. Indeed, a recent report describes the use of Ni-NTA in an improved SlyD purification protocol and also establishes SlyD as "the only E. coli protein capable of contaminating denaturing IMAC-based procedures."(2)
IMAC resins have highly specific affinity for His-tags, which allows for single-step purification to near homogeneity. However, we have observed a propensity for significant SlyD contamination when using Ni-NTA, particularly to purify proteins whose genes are not well expressed in E. coli hosts. Even at fairly high levels of production typical samples of His-tagged Salmonella FliI produced in E. coli strain BL21(DE3)pLysS by induction for four hours with 0.2 mM IPTG at 30 C, and purified by Ni-NTA chromatography, reveal substantial contamination by a 27 kDa band (Figure 2). N-terminal amino acid sequencing positively identifies this contaminant as SlyD (Guoyong Li, unpublished data).
In order to compare Ni-NTA and TALON Resin with respect to SlyD binding, a side-by-side experiment was undertaken. BL21(DE3)pLysS was transformed with pET19b (Novagen, Madison, Wis.) containing no insert. An overnight culture in Luria-Bertani media supplemented with 100 mg/ml ampicillin was subcultured and grown at 30 C to an OD600 of 0.4. Cells were induced with 0.2 mM IPTG and growth was continued for 4 hours. Cells were then harvested by centrifugation and pellets were stored at -85 C overnight.
Cell pellets of equal volume from the same culture were thawed and resuspended in either 50 mM phosphate buffer (pH 8.0), 500 mM NaCl, 10 mM imidazole (native buffer) or the same with 6 M urea (denaturing buffer). Suspensions were sonicated and subjected to centrifugation at 100,000 3g for 45 min. Supernatants (cleared lysates) were further divided into two equal fractions and applied to 250 µl Ni-NTA or BD TALON columns. The columns were washed with 20 volumes of native or denaturing buffer plus 20 mM imidazole. Bound protein was then eluted in three fractions of 250 µl with native or denaturing buffer containing 250 mM imidazole. The fractions were analyzed by SDS-PAGE (Figure 3).
Under both denaturing (Figure 3A) and native (Figure 3B) conditions, Ni-NTA bound substantial quantities of the 27 kDa contaminant that corresponds to SlyD (6 mg/l of contaminating proteins under native conditions and 2.5 mg/l of SlyD under denaturing conditions). Other contaminating bands were also present in the native Ni-NTA samples. No contamination by SlyD or any other protein was detected with BD TALON Resin under either condition.
Lysates containing large amounts of His-tagged proteins are usually amenable to Ni-NTA purification because the His-tagged protein outcompetes SlyD for resin binding. High purity samples of His-tagged proteins can be achieved with Ni-NTA by saturating the resin, but this is wasteful and can be difficult when working with genes that are not well expressed. When TALON Resin is used, even under conditions of low (or, as in the experiment above, absent) production of His-tagged proteins, no SlyD contamination is evident. Thus, additional chromatographic or other steps are not necessary to achieve homogeneity.
About the authorsThe authors work in the Department of Molecular Biophysics and Biochemistry at Yale University, New Haven, Conn. More information about BD Talon Resin is available from: BD Biosciences.Use InfoLINK 4A1510 or Call 800-287-0633
References
1. Hottenrott, S. et. al. The Escherichia coli SlyD is a metal-ion regulated peptidyl-prolyl cis/trans-isomerase. J. Biol. Chem. 272:15697-15701 (1997).
2. Mukherjee, S. et. al. Single-step purification of a protein-folding catalyst, the SlyD peptidyl prolyl isomerase (PPI), from cytoplasmic extracts of Escherichia coli. Biotechnol. Appl. Biochem. 37:183-186 (2003).
3. Wülfing, C. et. al. A. An Escherichia coli protein consisting of a domain homologous to FK506-binding proteins (FKBP) and a new metal binding motif. J. Biol. Chem. 269:2895-2901 (1994).
Figure 1: Figure 1. E. coli SlyD sequence. The metal-binding histidine-rich C-terminal domain is shown in bold with histidine residues underlined. Figure 2: SlyD contamination in a preparation of His-tagged FliI purified by Ni-NTA affinity chromatography. Three fractions of FliI eluted by 250 mM imidazole are shown. Positions of molecular weight standards are shown at left. Figure 3: FSlyD binds to Ni-NTA but not BD TALON columns. Panel A. Denaturing conditions. Panel B. Native conditions. Lane 1: cleared lysate. Lane 2: Ni-NTA final wash. Lanes 3-5: Ni-NTA elution fractions. Lane 6: BD TALON Resin final wash. Lanes 7-9: BD TALON Resin elution fractions. |
SlyD, an E. coli prolyl cis/trans-isomerase, binds and is regulated by divalent metal ions.(1) It contains a C-terminal histidine-rich metal binding domain (Figure 1). SlyD possesses a high affinity for nickel ions,(3) presenting a potential problem for Ni-based IMAC. Indeed, a recent report describes the use of Ni-NTA in an improved SlyD purification protocol and also establishes SlyD as "the only E. coli protein capable of contaminating denaturing IMAC-based procedures."(2)
IMAC resins have highly specific affinity for His-tags, which allows for single-step purification to near homogeneity. However, we have observed a propensity for significant SlyD contamination when using Ni-NTA, particularly to purify proteins whose genes are not well expressed in E. coli hosts. Even at fairly high levels of production typical samples of His-tagged Salmonella FliI produced in E. coli strain BL21(DE3)pLysS by induction for four hours with 0.2 mM IPTG at 30 C, and purified by Ni-NTA chromatography, reveal substantial contamination by a 27 kDa band (Figure 2). N-terminal amino acid sequencing positively identifies this contaminant as SlyD (Guoyong Li, unpublished data).
In order to compare Ni-NTA and TALON Resin with respect to SlyD binding, a side-by-side experiment was undertaken. BL21(DE3)pLysS was transformed with pET19b (Novagen, Madison, Wis.) containing no insert. An overnight culture in Luria-Bertani media supplemented with 100 mg/ml ampicillin was subcultured and grown at 30 C to an OD600 of 0.4. Cells were induced with 0.2 mM IPTG and growth was continued for 4 hours. Cells were then harvested by centrifugation and pellets were stored at -85 C overnight.
Cell pellets of equal volume from the same culture were thawed and resuspended in either 50 mM phosphate buffer (pH 8.0), 500 mM NaCl, 10 mM imidazole (native buffer) or the same with 6 M urea (denaturing buffer). Suspensions were sonicated and subjected to centrifugation at 100,000 3g for 45 min. Supernatants (cleared lysates) were further divided into two equal fractions and applied to 250 µl Ni-NTA or BD TALON columns. The columns were washed with 20 volumes of native or denaturing buffer plus 20 mM imidazole. Bound protein was then eluted in three fractions of 250 µl with native or denaturing buffer containing 250 mM imidazole. The fractions were analyzed by SDS-PAGE (Figure 3).
Under both denaturing (Figure 3A) and native (Figure 3B) conditions, Ni-NTA bound substantial quantities of the 27 kDa contaminant that corresponds to SlyD (6 mg/l of contaminating proteins under native conditions and 2.5 mg/l of SlyD under denaturing conditions). Other contaminating bands were also present in the native Ni-NTA samples. No contamination by SlyD or any other protein was detected with BD TALON Resin under either condition.
Lysates containing large amounts of His-tagged proteins are usually amenable to Ni-NTA purification because the His-tagged protein outcompetes SlyD for resin binding. High purity samples of His-tagged proteins can be achieved with Ni-NTA by saturating the resin, but this is wasteful and can be difficult when working with genes that are not well expressed. When TALON Resin is used, even under conditions of low (or, as in the experiment above, absent) production of His-tagged proteins, no SlyD contamination is evident. Thus, additional chromatographic or other steps are not necessary to achieve homogeneity.
About the authorsThe authors work in the Department of Molecular Biophysics and Biochemistry at Yale University, New Haven, Conn. More information about BD Talon Resin is available from: BD Biosciences.Use InfoLINK 4A1510 or Call 800-287-0633
References
1. Hottenrott, S. et. al. The Escherichia coli SlyD is a metal-ion regulated peptidyl-prolyl cis/trans-isomerase. J. Biol. Chem. 272:15697-15701 (1997).
2. Mukherjee, S. et. al. Single-step purification of a protein-folding catalyst, the SlyD peptidyl prolyl isomerase (PPI), from cytoplasmic extracts of Escherichia coli. Biotechnol. Appl. Biochem. 37:183-186 (2003).
3. Wülfing, C. et. al. A. An Escherichia coli protein consisting of a domain homologous to FK506-binding proteins (FKBP) and a new metal binding motif. J. Biol. Chem. 269:2895-2901 (1994).
