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Purification of the CaaX-modified, dynamin-related large GTPase hGBP1 by coexpression with farnesyltransferase.

Fres JM, Müller S, Praefcke GJ - J. Lipid Res. (2010)

Bottom Line: The C-terminal modification regulates the cellular localization and biological activity of isoprenylated proteins.We have established a strategy to produce and purify recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase.Farnesylated hGBP1 displays altered GTPase activity and is able to interact with liposomes in the activated state.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine Cologne, Institute for Genetics, Zülpicher Strasse 47, 50674 Köln, Germany.

ABSTRACT
Over a hundred proteins in eukaryotic cells carry a C-terminal CaaX box sequence, which targets them for posttranslational isoprenylation of the cysteine residue. This modification, catalyzed by either farnesyl or geranylgeranyl transferase, converts them into peripheral membrane proteins. Isoprenylation is usually followed by proteolytic cleavage of the aaX tripeptide and methylation of the carboxyl group of the newly exposed isoprenylcysteine. The C-terminal modification regulates the cellular localization and biological activity of isoprenylated proteins. We have established a strategy to produce and purify recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase. Our system is based on the coexpression of hGBP1 with the two subunits of human farnesyltransferase in Escherichia coli and a chromatographic separation of farnesylated and unmodified protein. Farnesylated hGBP1 displays altered GTPase activity and is able to interact with liposomes in the activated state.

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Purification of farnesylated hGBP1 from coexpression with FTase in E. coli. A: Purification scheme for farnesylated proteins. hGBP1 was expressed with a MRGSHis6-tag in an E. coli coexpression system. The purification started with Nickel-NTA Sepharose affinity chromatography. HIC allowed to separate posttranslationally lipid modified protein from the unmodified form, and the final purification step was size exclusion chromatography. B: Elution profile of hGBP1 from a preparative HIC using Butyl Sepharose High Performance (GE Healthcare) with an ammonium sulfate gradient from 0.5 M to 0 M. The line indicates the decreasing salt gradient. C: Preparative size exclusion chromatography profiles of unmodified and farnesylated hGBP1 indicating a monomeric state of both forms. D: SDS-PAGE of pooled samples obtained from each of the purification steps stained with Coomassie., L, lysate, S, supernatant. 1, Pool after Nickel-NTA Sepharose affinity chromatography; 2, farnesylated hGBP1 after HIC; 3, unmodified hGBP1 and FTase after HIC; 4, farnesylated hGBP1 after size exclusion chromatography; and 5, unmodified hGBP1 after size exclusion chromatography.
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fig1: Purification of farnesylated hGBP1 from coexpression with FTase in E. coli. A: Purification scheme for farnesylated proteins. hGBP1 was expressed with a MRGSHis6-tag in an E. coli coexpression system. The purification started with Nickel-NTA Sepharose affinity chromatography. HIC allowed to separate posttranslationally lipid modified protein from the unmodified form, and the final purification step was size exclusion chromatography. B: Elution profile of hGBP1 from a preparative HIC using Butyl Sepharose High Performance (GE Healthcare) with an ammonium sulfate gradient from 0.5 M to 0 M. The line indicates the decreasing salt gradient. C: Preparative size exclusion chromatography profiles of unmodified and farnesylated hGBP1 indicating a monomeric state of both forms. D: SDS-PAGE of pooled samples obtained from each of the purification steps stained with Coomassie., L, lysate, S, supernatant. 1, Pool after Nickel-NTA Sepharose affinity chromatography; 2, farnesylated hGBP1 after HIC; 3, unmodified hGBP1 and FTase after HIC; 4, farnesylated hGBP1 after size exclusion chromatography; and 5, unmodified hGBP1 after size exclusion chromatography.

Mentions: Farnesylated hGBP1 was produced by coexpression with human FTase α and β in E. coli, which contain farnesylpyrophosphate as a precursor for ubiquinone and heme biosynthesis. The purification procedure is shown in Fig. 1A. After Nickel-NTA Sepharose affinity purification, the mixture contained His6-tagged hGBP1 as well as His6-tagged-FTase α and untagged FTase β (Fig. 1D). Mass spectrometric analysis revealed that the farnesylation was not quantitative (data not shown) and that the elution contained a mixture of farnesylated and unmodified hGBP1. The small differences in their biophysical properties prevented a separation of these two forms by ion-exchange, size exclusion chromatography, or isoelectric focusing (data not shown). Baseline purification of the farnesylated protein was made possible using a new hydrophobic interaction chromatography (HIC) resin, Butyl Sepharose HP (Fig. 1B). MALDI analysis showed no sign of unmodified protein in the final product and also no farnesylated protein in the pool of unmodified protein (Fig. 2). Furthermore, the copurified FTase (see Fig. 1D) was also removed by the size exclusion chromatography purification step (Fig. 1C). Using this method, we obtained approximately 60 mg (20 mg farnesylated/40 mg unmodified) of pure hGBP1 from 4l of bacterial culture.


Purification of the CaaX-modified, dynamin-related large GTPase hGBP1 by coexpression with farnesyltransferase.

Fres JM, Müller S, Praefcke GJ - J. Lipid Res. (2010)

Purification of farnesylated hGBP1 from coexpression with FTase in E. coli. A: Purification scheme for farnesylated proteins. hGBP1 was expressed with a MRGSHis6-tag in an E. coli coexpression system. The purification started with Nickel-NTA Sepharose affinity chromatography. HIC allowed to separate posttranslationally lipid modified protein from the unmodified form, and the final purification step was size exclusion chromatography. B: Elution profile of hGBP1 from a preparative HIC using Butyl Sepharose High Performance (GE Healthcare) with an ammonium sulfate gradient from 0.5 M to 0 M. The line indicates the decreasing salt gradient. C: Preparative size exclusion chromatography profiles of unmodified and farnesylated hGBP1 indicating a monomeric state of both forms. D: SDS-PAGE of pooled samples obtained from each of the purification steps stained with Coomassie., L, lysate, S, supernatant. 1, Pool after Nickel-NTA Sepharose affinity chromatography; 2, farnesylated hGBP1 after HIC; 3, unmodified hGBP1 and FTase after HIC; 4, farnesylated hGBP1 after size exclusion chromatography; and 5, unmodified hGBP1 after size exclusion chromatography.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2903825&req=5

fig1: Purification of farnesylated hGBP1 from coexpression with FTase in E. coli. A: Purification scheme for farnesylated proteins. hGBP1 was expressed with a MRGSHis6-tag in an E. coli coexpression system. The purification started with Nickel-NTA Sepharose affinity chromatography. HIC allowed to separate posttranslationally lipid modified protein from the unmodified form, and the final purification step was size exclusion chromatography. B: Elution profile of hGBP1 from a preparative HIC using Butyl Sepharose High Performance (GE Healthcare) with an ammonium sulfate gradient from 0.5 M to 0 M. The line indicates the decreasing salt gradient. C: Preparative size exclusion chromatography profiles of unmodified and farnesylated hGBP1 indicating a monomeric state of both forms. D: SDS-PAGE of pooled samples obtained from each of the purification steps stained with Coomassie., L, lysate, S, supernatant. 1, Pool after Nickel-NTA Sepharose affinity chromatography; 2, farnesylated hGBP1 after HIC; 3, unmodified hGBP1 and FTase after HIC; 4, farnesylated hGBP1 after size exclusion chromatography; and 5, unmodified hGBP1 after size exclusion chromatography.
Mentions: Farnesylated hGBP1 was produced by coexpression with human FTase α and β in E. coli, which contain farnesylpyrophosphate as a precursor for ubiquinone and heme biosynthesis. The purification procedure is shown in Fig. 1A. After Nickel-NTA Sepharose affinity purification, the mixture contained His6-tagged hGBP1 as well as His6-tagged-FTase α and untagged FTase β (Fig. 1D). Mass spectrometric analysis revealed that the farnesylation was not quantitative (data not shown) and that the elution contained a mixture of farnesylated and unmodified hGBP1. The small differences in their biophysical properties prevented a separation of these two forms by ion-exchange, size exclusion chromatography, or isoelectric focusing (data not shown). Baseline purification of the farnesylated protein was made possible using a new hydrophobic interaction chromatography (HIC) resin, Butyl Sepharose HP (Fig. 1B). MALDI analysis showed no sign of unmodified protein in the final product and also no farnesylated protein in the pool of unmodified protein (Fig. 2). Furthermore, the copurified FTase (see Fig. 1D) was also removed by the size exclusion chromatography purification step (Fig. 1C). Using this method, we obtained approximately 60 mg (20 mg farnesylated/40 mg unmodified) of pure hGBP1 from 4l of bacterial culture.

Bottom Line: The C-terminal modification regulates the cellular localization and biological activity of isoprenylated proteins.We have established a strategy to produce and purify recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase.Farnesylated hGBP1 displays altered GTPase activity and is able to interact with liposomes in the activated state.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine Cologne, Institute for Genetics, Zülpicher Strasse 47, 50674 Köln, Germany.

ABSTRACT
Over a hundred proteins in eukaryotic cells carry a C-terminal CaaX box sequence, which targets them for posttranslational isoprenylation of the cysteine residue. This modification, catalyzed by either farnesyl or geranylgeranyl transferase, converts them into peripheral membrane proteins. Isoprenylation is usually followed by proteolytic cleavage of the aaX tripeptide and methylation of the carboxyl group of the newly exposed isoprenylcysteine. The C-terminal modification regulates the cellular localization and biological activity of isoprenylated proteins. We have established a strategy to produce and purify recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase. Our system is based on the coexpression of hGBP1 with the two subunits of human farnesyltransferase in Escherichia coli and a chromatographic separation of farnesylated and unmodified protein. Farnesylated hGBP1 displays altered GTPase activity and is able to interact with liposomes in the activated state.

Show MeSH
Related in: MedlinePlus