Protein Purification Strategies Research Articles

Affinity Capture of TAP-tagged Protein Complexes: Affinity Purification Step 2

INTRODUCTIONOne approach to identifying protein–protein interactions is the biochemical purification of a target protein from cells or tissues under nondenaturing conditions, followed by the mass spectrometric identification of the components of the purified protein complex. The combination of highly specific protein purification strategies and mass spectrometry has proven to be a very successful approach for identifying protein–protein interactions. The tandem affinity purification (TAP) affinity tag and purification method allows efficient recovery of proteins present at low cellular concentrations under native conditions. Expressing the target protein at its natural levels avoids the assembly of overexpressed proteins into nonphysiological complexes. This protocol describes calmodulin affinity capture of the TAP-tagged complexes after immunoglobulin G (IgG) affinity purification has been performed. These complexes can then be analyzed by methods such as high-sensitivity liquid chromatography coupled with tandem mass spectrometry and/or multidimensional protein identification technology (MudPIT) analysis.

Making Yeast Cell Extracts for Purifying TAP-tagged Protein Complexes

INTRODUCTIONOne approach to identifying protein–protein interactions is the biochemical purification of a target protein from cells or tissues under nondenaturing conditions followed by the mass spectrometric identification of the components of the purified protein complex. The combination of highly specific protein purification strategies and mass spectrometry has proven to be a very successful approach for identifying protein–protein interactions. The tandem affinity purification (TAP) affinity tag and purification method allows efficient recovery of proteins present at low cellular concentrations under native conditions. Expressing the target protein at its natural levels avoids the assembly of overexpressed proteins into nonphysiological complexes. This protocol describes the preparation of extract from TAP-tagged yeast cells for use in purifying TAP-tagged protein complexes.

IgG Affinity Capture of TAP-tagged Protein Complexes from Cell Extracts: Affinity Purification Step 1

INTRODUCTIONOne approach to identifying protein–protein interactions is the biochemical purification of a target protein from cells or tissues under nondenaturing conditions, followed by the mass spectrometric identification of the components of the purified protein complex. The combination of highly specific protein purification strategies and mass spectrometry has proven to be a very successful approach for identifying protein–protein interactions. The tandem affinity purification (TAP) affinity tag and purification method allows efficient recovery of proteins present at low cellular concentrations under native conditions. Expressing the target protein at its natural levels avoids the assembly of overexpressed proteins into nonphysiological complexes. This protocol describes the immunoglobulin G (IgG) affinity capture of TAP-tagged complexes. This procedure is followed by calmodulin affinity purification.

The development, characterization, and demonstration of a novel strategy for purification of recombinant proteins expressed in plants

Plants have attracted increasing attention as an expression platform for the production of pharmaceutical proteins due to its unlimited scalability and low cost potential. However, compared to other expression systems, plants accumulate relatively low levels of foreign proteins, thus necessitating the development of efficient systems for purification of foreign proteins from plant tissues. We have developed a novel strategy for purification of recombinant proteins expressed in plants, based on genetic fusion to soybean agglutinin (SBA), a homotetrameric lectin that binds to N-acetyl-D-galactosamine. Previously it was shown that high purity SBA could be recovered from soybean with an efficiency of greater than 90% following one-step purification using N-acetyl-D-galactosamine-agar columns. We constructed an SBA fusion protein containing the reporter green fluorescent protein (GFP) and transiently expressed it in N. benthamiana plants. We achieved over 2.5% of TSP accumulation in leaves of N. benthamiana. Confocal microscopic analysis demonstrated in vivo activity of the fused GFP partner. Importantly, high purity rSBA-GFP was recovered from crude leaf extract with ~90% yield via one-step purification on N-acetyl-D-galactosamine-agar columns, and the purified fusion protein was able to induce the agglutination of rabbit red blood cells. Combined with this, tetrameric assembly of the fusion protein was demonstrated via western blotting. In addition, rSBA-GFP retained its GFP signal on agglutinated red blood cells, demonstrating the feasibility of using rSBA-GFP for discrimination of cells that bear the ligand glycan on their surface. This work validates SBA as an effective affinity tag for simple and rapid purification of genetically fused proteins.

Microbial polyhydroxyalkanote synthesis repression protein PhaR as an affinity tag for recombinant protein purification

BackgroundPhaR which is a repressor protein for microbial polyhydroxyalkanoates (PHA) biosynthesis, is able to attach to bacterial PHA granules in vivo, was developed as an affinity tag for in vitro protein purification. Fusion of PhaR-tagged self-cleavable Ssp DnaB intein to the N-terminus of a target protein allowed protein purification with a pH and temperature shift. During the process, the target protein was released to the supernatant while PhaR-tagged intein was still immobilized on the PHA nanoparticles which were then separated by centrifugation.ResultsFusion protein PhaR-intein-target protein was expressed in recombinant Escherichia coli. The cell lysates after sonication and centrifugation were collected and then incubated with PHA nanoparticles to allow sufficient absorption onto the PHA nanoparticles. After several washing processes, self-cleavage of intein was triggered by pH and temperature shift. As a result, the target protein was released from the particles and purified after centrifugation. As target proteins, enhanced green fluorescent protein (EGFP), maltose binding protein (MBP) and β-galactosidase (lacZ), were successfully purified using the PhaR based protein purification method.ConclusionThe successful purification of EGFP, MBP and LacZ indicated the feasibility of this PhaR based in vitro purification system. Moreover, the elements used in this system can be easily obtained and prepared by users themselves, so they can set up a simple protein purification strategy by themselves according to the PhaR method, which provides another choice instead of expensive commercial protein purification systems.

Surface functionalization of polyketal microparticles with nitrilotriacetic acid–nickel complexes for efficient protein capture and delivery

Microparticle drug delivery systems have been used for over 20 years to deliver a variety of drugs and therapeutics. However, effective microencapsulation of proteins has been limited by low encapsulation efficiencies, large required amounts of protein, and risk of protein denaturation. In this work, we have adapted a widely used immobilized metal affinity protein purification strategy to non-covalently attach proteins to the surface of microparticles. Polyketal microparticles were surface modified with nitrilotriacetic acid–nickel complexes which have a high affinity for sequential histidine tags on proteins. We demonstrate that this high affinity interaction can efficiently capture proteins from dilute solutions with little risk of protein denaturation. Proteins that bound to the Ni–NTA complex retain activity and can diffuse away from the microparticles to activate cells from a distance. In addition, this surface modification can also be used for microparticle targeting by tethering cell-specific ligands to the surface of the particles, using VE-Cadherin and endothelial cells as a model. In summary, we show that immobilized metal affinity strategies have the potential to improve targeting and protein delivery via degradable polymer microparticles.

Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (C(in)) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (C(out)) to C(in) ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C(out) topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescence-detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C(out) topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging.

Assessment of a Plantibody HB-01 Purification Strategy at Different Scales

Transgenic plant investigations focus on fine-regulation of the recombinant protein expression and purification strategies. In this article, preliminary experiments were done at analytical-scale to decide the best plantibody HB-01 purification strategy. Once it was assumed, the purification efficiency was assessed at different scales (10–600 kg of biomass). The plantibody purity measured by SDS-PAGE and LC-GF was over 90%, yielding 9.9 ± 6.2–18.6 ± 0.9 mg plantibody kg−1 of biomass and 39.9 ± 7.9–48.7 ± 2.1% of recovery. Significant differences were not observed among these parameters at these scales. Plant DNA contents were <3.3 ng mg−1 of plantibody, which is considered very low for the plantibody HB-01 application in the hepatitis B vaccine production.

RNA biophysics has come of age

RNA biophysics has come of age

Simultaneous refolding and purification of a recombinant lipase with an intein tag by affinity precipitation with chitosan

A strategy for simultaneous purification and refolding of proteins overexpressed with an intein tag is described. A recombinant lipase overexpressed in Escherichia coli ER2566 with the intein tag and obtained as inclusion bodies was solubilized in buffer containing 8 M urea or cetyltrimethylammonium bromide. The solubilized lipase was precipitated with chitosan and the affinity complex of the polymer with the fusion protein was obtained. The intein tag was cleaved with dithiothreitol and the refolded lipase was obtained in active form. Activity recovery of 80% was observed and the enzyme had a specific activity of 2965 units/mg. The purified lipase showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purity and activity recovery were comparable with that of the preparation obtained by using the commercial kit which utilizes chromatography on chitin beads. The purified and refolded lipase was characterized by fluorescence and CD spectroscopy.

Purification of recombinant GFP produced by Agrobacterium-mediated transient expression in Nicotiana excelsior

Green fluorescent protein (GFP) is commonly used as a reporter protein in a wide range of biological experiments. The efficient protocol of Agrobacterium-mediated transient expression in Nicotiana excelsior was applied for quick preparative production of recombinant GFP. The protein purification scheme has been developed and included ammonium sulfate precipitation and Q-sepharose anion-exchange chromatography. It results in obtaining of a fraction with about 85% GFP homogeneity and the protein yield of about 75%.

Automated production of recombinant human proteins as resource for proteome research

BackgroundAn arbitrary set of 96 human proteins was selected and tested to set-up a fully automated protein production strategy, covering all steps from DNA preparation to protein purification and analysis. The target proteins are encoded by functionally uncharacterized open reading frames (ORF) identified by the German cDNA consortium. Fusion proteins were produced in E. coli with four different fusion tags and tested in five different purification strategies depending on the respective fusion tag. The automated strategy relies on standard liquid handling and clone picking equipment.ResultsA robust automated strategy for the production of recombinant human proteins in E. coli was established based on a set of four different protein expression vectors resulting in NusA/His, MBP/His, GST and His-tagged proteins. The yield of soluble fusion protein was correlated with the induction temperature and the respective fusion tag. NusA/His and MBP/His fusion proteins are best expressed at low temperature (25°C), whereas the yield of soluble GST fusion proteins was higher when protein expression was induced at elevated temperature. In contrast, the induction of soluble His-tagged fusion proteins was independent of the temperature. Amylose was not found useful for affinity-purification of MBP/His fusion proteins in a high-throughput setting, and metal chelating chromatography is recommended instead.ConclusionSoluble fusion proteins can be produced in E. coli in sufficient qualities and μg/ml culture quantities for downstream applications like microarray-based assays, and studies on protein-protein interactions employing a fully automated protein expression and purification strategy. Future applications might include the optimization of experimental conditions for the large-scale production of soluble recombinant proteins from libraries of open reading frames.

Production of epoxide hydrolases in batch fermentations of Botryosphaeria rhodina

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.

Orthogonal protein purification—Expanding the repertoire of GST fusion systems

We have previously developed a labeling scheme that can be used to site-specifically link human glutathione transferases (hGSTs) from the alpha class to chemical entities such as fluorophores and aldehydes. The reagents are in-house synthesized derivatives of glutathione (GS-derivatives). We have focused on a lysine mutant of hGST A1:A216K. In this study, we wanted to utilize these findings and improve on protein purification schemes that are using GSTs as fusion partners. We have used random mutagenesis to scramble the hydrophobic binding site of A216K through mutations at position M208 and isolated a library of 11 A216K/M208X mutants. All mutants were easily expressed and purified and retained all or parts of the catalytic properties of the parent GST. The mutants were stable over several days at room temperature. The A216K/M208X mutants could be site-specifically labeled using our designed fluorescent reagents. Furthermore, reaction with an aldehyde-containing reagent termed GS-Al results in site-specific introduction of an orthogonal handle for subsequent conjugation with aldehyde-reactive probes. Labeling with coumarin results in a fluorescent protein-conjugate that can bind glutathione (GSH) derivatives for subsequent affinity purification. The K d for S-hexyl-GSH of coumarin-labeled A216K was measured to be 2.5 μM. The candidate proteins A216K and A216K/M208F could be purified in high yield in a one-step procedure through affinity chromatography (Glutathione Sepharose™ 4B). The proteins can readily be perceived as improved GST fusion partners.

High‐throughput protein purification under denaturating conditions by the use of cation exchange chromatography

A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.

Hydrodynamic Studies on the Quaternary Structure of Recombinant Mouse Purβ

Purbeta is a gene regulatory factor belonging to a family of highly conserved nucleic acid-binding proteins related by their ability to preferentially bind single-stranded DNA or RNA sequences rich in purine nucleotides. In conjunction with Puralpha, Purbeta has been implicated in transcriptional and translational repression of genes encoding contractile proteins found in the heart and vasculature. Although several models of sequence-specific DNA recognition, strand separation, and activator inhibition by oligomeric Puralpha and Purbeta have been proposed, it is currently unclear whether protein-protein interaction is a prerequisite to, or a consequence of nucleic acid binding. In this study, a recombinant protein purification scheme was devised to yield homogenous mouse Purbeta devoid of nucleic acid. Recombinant Purbeta was then subjected to light scattering and analytical ultracentrifugation analyses to assess the size, shape, and oligomeric state of the purified protein in solution. Results of laser light scattering and sedimentation velocity experiments indicated that Purbeta reversibly self-associates in the absence of nucleic acid. Both approaches independently showed that the hydrodynamic shape of the Purbeta homodimer is markedly asymmetric and non-spherical. Sedimentation velocity analyses indicated that dimeric Purbeta has a sedimentation coefficient of 3.96 Svedberg, a frictional coefficient ratio (f/f(0)) of 1.60, and a hydrodynamic radius of 4.43 nm. These values were consistent with those determined by independent dynamic light scattering studies. Sedimentation equilibrium analyses confirmed that Purbeta self-associates in a reversible monomer-dimer equilibrium characterized by a K(d) = 1.13 +/- 0.27 microm.

Transactivation and signaling functions of Tat are not correlated: biological and immunological characterization of HIV-1 subtype-C Tat protein

BackgroundOf the diverse subtypes of Human Immunodeficiency Virus Type-1 (HIV-1), subtype-C strains cause a large majority of infections worldwide. The reasons for the global dominance of HIV-1 subtype-C infections are not completely understood. Tat, being critical for viral infectivity and pathogenesis, may differentially modulate pathogenic properties of the viral subtypes. Biochemical studies on Tat are hampered by the limitations of the current purification protocols. Tat purified using standard protocols often is competent for transactivation activity but defective for a variety of other biological functions. Keeping this limitation in view, we developed an efficient protein purification strategy for Tat.ResultsTat proteins obtained using the novel strategy described here were free of contaminants and retained biological functions as evaluated in a range of assays including the induction of cytokines, upregulation of chemokine coreceptor, transactivation of the viral promoter and rescue of a Tat-defective virus. Given the highly unstable nature of Tat, we evaluated the effect of the storage conditions on the biological function of Tat following purification. Tat stored in a lyophilized form retained complete biological activity regardless of the storage temperature. To understand if variations in the primary structure of Tat could influence the secondary structure of the protein and consequently its biological functions, we determined the CD spectra of subtype-C and -B Tat proteins. We demonstrate that subtype-C Tat may have a relatively higher ordered structure and be less flexible than subtype-B Tat. We show that subtype-C Tat as a protein, but not as a DNA expression vector, was consistently inferior to subtype-B Tat in a variety of biological assays. Furthermore, using ELISA, we evaluated the anti-Tat antibody titers in a large number of primary clinical samples (n = 200) collected from all four southern Indian states. Our analysis of the Indian populations demonstrated that Tat is non-immunodominant and that a large variation exists in the antigen-specific antibody titers.ConclusionOur report not only describes a simple protein purification strategy for Tat but also demonstrates important structural and functional differences between subtype-B and -C Tat proteins. Furthermore, this is the first report of protein purification and characterization of subtype-C Tat.

Structure analysis of light harvesting complexes from the dinoflagellateAmphidinium carterae

Dinoflagellates make use of a two component light harvesting system in order to increase energy inflow to the two photosystems. One is the membrane bound light harvesting complex LHC, which shows homology to higher plant CAB-proteins, but contains chl c instead of chl b [1]. The other complex is the unique, water soluble peridinin-chlorophyll a-protein (PCP) predicted to be located in the thylakoid luminal space [2, 3]. This complex occurs in different isoforms with the mainform showing a pI of 7.5 (MFPCP). Both complexes excessively utilize the carotenoid peridinin for light harvesting. The structure of the main form of PCP has been solved at a resolution of 2A. We were able to produce a new crystal form of PCP with spacegroup P42212. The structure has been solved by MR using the MFPCP as a search model and has been refined at a resolution of 2.9A. While we observe a different packing arrangement in the crystal the protein still forms the trimer found in other spacegroups. The LHC of the dinoflagellate A.carterae has been extensively studied spectroscopically, but earlier attempts to determine its three dimensional structure by X-ray crystallography only yielded very small crystals too small for further characterization [4]. Here we report an optimized protein purification scheme, yielding reproducibly large amounts of protein for crystallization. We were able to identify new crystallization conditions, under which we could grow crystals of sizes up to 600x50x10μm 3 in one to three month. Initial data have been collected to 3.5A resolution. A preliminary crystallographic characterization will be presented.

Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin

The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed in Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37 °C or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fusion protein that prevent the second and third steps of protein splicing. As a result, the intein fusion protein can facilitate temperature-dependent formation of a thioester linkage between the N-extein and intein. This thioester is susceptible to in vitro hydrolysis or thiolysis at temperatures of 40 °C or higher, and we have exploited this activity to generate a temperature-dependent protein purification scheme. Protein purification using this intein does not require the addition of exogenous thiols and is compatible with the use of immobilized metal affinity chromatography. The identity of the C-terminal residue of the N-extein has less influence on the cleavage reaction than in current purification systems in terms of premature in vivo cleavage and is complementary to current systems in terms of efficient in vitro cleavage.

Surface-Assisted Delivery of Fluorescent Groups to hGST A1-1 and a Lysine Mutant

Human glutathione transferase (hGST) A1-1 and a lysine mutant (A216K) can both be rapidly and site-specifically acylated on Y9 and K216, respectively, using a range of thiolesters of glutathione (GS-thiolesters) as modifying reagents. The present investigation was aimed at developing a method with which to deliver a fluorescent acyl group from a solid support under conditions compatible with standard protein purification schemes. A number of fluorescent GS-thiolesters with modified peptide backbones were therefore prepared and tested for reactivity toward hGST A1-1 and the A216K mutant. Substitutions at the alpha-NH2 part of the glutathione backbone were not tolerated by the proteins. However, two fluorescent reagents that carry a biotin moiety at the C-terminal part of glutathione were found through MALDI-MS experiments to react in solution with Y9 of the wild-type protein and one reagent with K216 of A216K. The reaction can take place in the presence of glutathione and even in a crude E. coli lysate of cells expressing A216K. Delivery of the fluorescent group to Y9 or K216 was possible using NeutrAvidin (NA) beads that had been preincubated with biotinylated reagent. Alternatively, excess reagent can be removed by a brief incubation with NA beads. We have thus now developed a system for protein labeling with easy removal of excess and used up low-molecular weight reagent. This strategy can conceivably be utilized in future protein purification and labeling experiments.