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- Materials and methods
The preparation of proteins for structural and functional analysis using the Escherichia coli expression system is often hampered by the formation of insoluble intracellular protein aggregates (inclusion bodies). Transferring those proteins into their native states by in vitro protein folding requires screening for the best buffer conditions and suitable additives. However, it is difficult to assess the success of such a screen if no biological assay is available. We established a fully automated folding screen and a system to detect folded protein that is based on analytical hydrophobic interaction chromatography and tryptophan fluorescence spectroscopy. The system was evaluated with two model enzymes (carbonic anhydrase II and malate dehydrogenase), and was successfully applied to the folding of the p22 subunit of human dynactin, which is expressed in inclusion bodies in E. coli. The described screen allows for high-throughput folding analysis of inclusion body proteins for structural and functional analyses.
Upon overexpression in Escherichia coli, a large proportion of proteins accumulates in insoluble, intracellular aggregates. These inclusion bodies contain high amounts of the target protein in relatively high purity. The structures of inclusion body proteins differ from their respective native conformations, for example, an increased level of nonnative β-sheets appears to be common among them (Fink 1998). Inclusion body protein, solubilized by denaturants or detergents, can be folded to the native conformation by transfer into an appropriate buffer, using dilution or dialysis. This process is in some cases prone to high losses due to misfolding and aggregation, but may be shifted to higher yields by “folding helpers” (e.g., polyethylene glycol, glycerol, detergents, arginine, etc.). Many other factors including pH, protein concentration, or the redox milieu can influence the success of folding of a particular protein. Protein folding of denaturant- or detergent-solubilized proteins has been extensively reviewed (Guise et al. 1996; Rudolph and Lilie 1996; Lilie et al. 1998; Misawa and Kumagai 1999; Clark 2001; Middelberg 2002).
Protein folding has also been achieved by binding the protein to a chromatographic resin in the unfolded state and subsequent washing with an appropriate buffer that contains no denaturant. Affinity resins like NiNTA sepharose (Zahn et al. 1997; Rehm et al. 2001) or heparin sepharose (Stempfer et al. 1996) have been used for poly-His-tagged or poly-Arg-tagged proteins, respectively. Compared to folding by dialysis or rapid dilution, this method has two advantages. Aggregation due to intermolecular interaction of partly folded species is prevented, and the protein is obtained in higher concentration after folding. However, an interference of the chromatographic support with the folding protein molecule can be detrimental, causing precipitation of the protein on the matrix.
A protein folding screen for identification of optimal folding conditions using rapid dilution or dialysis has been developed by Gouaux and colleagues (Chen and Gouaux 1997; Armstrong et al. 1999). Related screens have been used by others (Heiring and Muller 2001; Tobbell et al. 2002). This so-called fractional factorial screen is also commercially available (FoldIt Screen, Hampton Research) and consists of an initial screen of 16 conditions, in which 12 parameters (various additives, pH, protein concentration) are altered. Each parameter appears in eight of the 16 conditions. Therefore, a couple of parameters are varied and tested at the same time, instead of checking each parameter independently. Thus, it is an approach to screen a high number of parameters with a relatively small number of folding buffers.
When screening for the best conditions for protein folding, it is difficult to determine the yield of native protein obtained if no biological assay is available. Centrifugation or filtration can remove precipitates, but solubility cannot be used as a stringent criterion, because soluble microaggregates and partially folded intermediates are not distinguished from the natively folded protein. To prevent aggregation, low protein concentrations are commonly used in folding experiments. Therefore, sensitive methods are required for detection. Analytical gel filtration can be used (Chen and Gouaux 1997). However, protein concentrations are often too low to detect folded protein. In addition, the method is time-consuming. Another method applied is limited proteolysis (Heiring and Muller 2001). Partially folded intermediates are assumed to be more susceptible to subtilisin-induced proteolysis than native protein. Although this approach is very sensitive and does not require high sample purity, there are some disadvantages. Range-finding experiments for the protease concentration may be necessary. Additionally, false positives or false negatives can occur because soluble nonnative microaggregates might be equally or even less susceptible to proteolysis than native protein, thus leading to a misinterpretation of the results.
In this work, we describe a folding screen that is performed automatically using a pipetting robot. In contrast to a fractional factorial screen we chose a screening-strategy in which only one parameter is changed at a time. The tested conditions comprise folding by rapid dilution and, in the case of His-tagged proteins, folding on metal chelate resins.
Tryptophan fluorescence spectroscopy was evaluated as a monitor to detect folded protein, as unfolded conformations, folding intermediates, and native proteins are distinguishable in their respective spectra (Royer 1995). The wavelength of the fluorescence intensity maximum is shifted to smaller values (blue-shift) upon folding to the native state from the denaturant-unfolded protein. We tested whether the occurrence of a blue-shift is an indicator for folded protein in a folding screen.
Tryptophan residues are commonly more solvent exposed in unfolded or partially folded proteins than in native proteins. Solvent exposure usually causes quenching of tryptophan fluorescence. Therefore, we assumed that high fluorescence intensity is an indicator for folded protein. However, this assumption does not hold for all proteins. A few exceptions are known (e.g., γF-crystallin; Das and Liang 1998) where the tryptophan fluorescence is strongly quenched in the native conformation by histidines, disulfide bonds, or chromophores. Hence, fluorescence intensity should only be used in conjunction with other folding monitors.
For some proteins, the evaluation of a folding screen with tryptophan fluorescence might be difficult due to low signal intensity; other proteins do not contain any tryptophans. Therefore, we tried to find an alternative method and tested whether hydrophobic interaction chromatography (HIC) can be used as a monitor to detect folded protein. During HIC, proteins are separated due to differences of surface hydrophobicity. Therefore, HIC should discriminate the compact native protein from partially folded or misfolded species that usually expose more hydrophobic patches. Soluble aggregates, which are assumed to form via interaction of hydrophobic patches between folding intermediates (Fink 1998), should also be distinguished.
An evaluation of the efficiency of the automated folding screen with the described biophysical analysis system was performed by refolding two denatured model enzymes, bovine erythrocyte carbonic anhydrase II (CAB), and pig heart mitochondrial malate dehydrogenase (MDH), and correlating the obtained data with enzymatic activity. In vitro refolding of CAB is strongly hampered by aggregation. CAB has therefore been used as a model to study aggregation processes during refolding (Cleland and Wang 1990). MDH was shown to be a substrate of the GroEL/GroES folding system during in vitro refolding (Weber et al. 1998). In this study, the chaperones enhanced MDH refolding efficiency by more than a magnitude compared to refolding without GroEL/GroES.
Furthermore, we tested whether the developed system is suitable to determine conditions to fold the p22 subunit of human dynactin that is expressed as inclusion bodies in E. coli. Dynactin is a macromolecular complex that is an activator for cytoplasmic dynein-mediated transport (Gill et al. 1991). The first report on the smallest subunit, p22 (21 kD), was by Holzbaur and coworkers (Karki et al. 1998). In this study, the authors noted a novel localization for cytoplasmic dynein and dynactin at the cleavage furrow and the midbody of dividing cells. So far, no structural information of the dynactin subunits is known. For the p22 subunit, the closest structural homolog is the β-domain of streptokinase, with a sequence identity of 25%, covering 53% of the sequence of p22 dynactin. Therefore, we tried to obtain a preparation of pure and native p22 dynactin for structural analysis.
Materials and methods
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- Materials and methods
Secondary structure prediction of p22 dynactin was performed with PSIPRED (Department of Computer Science, University College London).
Bovine erythrocyte carbonic anhydrase II (CAB) was purchased from Sigma, pig heart malate dehydrogenase (MDH) from Merck. Protein concentrations were determined from the absorbance at 280 nm using the extinction coefficient calculated from the amino acid sequence (Mach et al. 1992). Absorbance was corrected for stray light according to the light-scattering theory (Tyndall effect, I(s) ∼ λ−4), with the assumption that no absorption due to protein chromophores occur above 320 nm (Levine and Federici 1982). For protein concentration determination of conditions 1–18 of refolded MDH, 250-μL aliquots were centrifuged at 25,000 × g for 30 min at 8°C, and supernatants were analyzed with the Bio-Rad Bradford Assay with native MDH as the standard. Arginine and CTAB present in conditions 19–22 are not compatible with this assay.
CAB and MDH were denatured for 5 h at room temperature. Conditions used were: (1) 6 mg/mL CAB in 20 mM Tris-HCl, pH 7.7, containing 6 M GdmHCl and 2 mM EDTA; and (2) 2.24 mg/mL MDH in 20 mM Tris-HCl, pH 7.7, containing 7 M GdmHCl and 10 mM DTT.
Expression of recombinant p22 dynactin in E. coli
p22 dynactin (GenBank accession AAC61280) was expressed at 37°C in E. coli SCS1 cells using the pQTEV expression vector (GenBank accession AY243506). SCS1 cells contained the Rosetta helper plasmid (Novagen). Proteins expressed from the pQTEV vector carried an N-terminal heptahistidine-tag (His-tag) followed by a TEV-protease cleavage site. A 100-mL overnight culture was prepared in 2xYT medium (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, pH 7.0), supplemented with 2% glucose, 100 μg/mL ampicillin, and 30 μg/mL chloramphenicol. This culture was transferred into 1.9 L of SB medium (12 g/L Bacto-tryptone, 24 g/L yeast extract, 0.4% [v/v] glycerol, 17 mM KH2PO4, 72 mM K2HPO4), supplemented with 20 μg/mL thiamine, 100 μg/mL ampicillin, and 30 μg/mL chloramphenicol. At an OD600 of 1.5, protein expression was induced for 4 h by adding IPTG to a final concentration of 1 mM. Cells were harvested by centrifugation (4000 × g for 20 min at 4°C) and washed with 20 mM Tris-HCl, pH 7.7, 150 mM NaCl. They were frozen in liquid N2 and stored at −80°C until use.
Inclusion body preparation and denaturing His-tag purification of p22 dynactin
Cells were suspended in ice-cold 20 mM Tris-HCl, pH 7.7, 300 mM NaCl, 2 mM DTT, 1 mM EDTA, before disruption by sonification. The crude extract was centrifuged at 25,000 × g for 45 min, 10°C, and the pellet was washed twice in 20 mM Tris-HCl, pH 7.7, 300 mM NaCl. The resulting pellet was dissolved in 20 mM Tris-HCl, pH 7.7, 6.6 M GdmHCl, 30 mM NaCl, 5 mM imidazole, 2 mM β-mercaptoethanol overnight under stirring at room temperature. The GdmHCl-insoluble material was removed by centrifugation (25,000 × g, 60 min) and the supernatant was applied to a 7.8-mL Ni-POROS20-column (Applied Biosystems), previously equilibrated in urea buffer (20 mM Tris-HCl, pH 7.7, 8 M urea, 30 mM NaCl, 5 mM imidazole, 2 mM β-mercaptoethanol). After washing with 15 column volumes of urea buffer, bound protein was eluted using urea buffer enriched with 300 mM imidazole. The protein concentration was adjusted to 5 mg/mL by dilution into urea buffer. EDTA and DTT was added to achieve final concentrations of 1 mM and 15 mM, respectively, followed by a 60-min incubation at room temperature to reduce cysteines. The obtained protein solution was used for rapid dilution screen conditions (Table 1). For screening conditions where p22 dynactin is immobilized on NiNTA agarose or Fractogel EMD Chelate during folding, EDTA, DTT, and imidazole had to be removed by a buffer exchange into urea buffer using a HiTrap desalting column (Amersham Bioscience). p22 dynactin (1.2 mg) was incubated for 20 min while shaking with 350 μL NiNTA agarose or 350 μL Fractogel EMD Chelate, loaded with Ni2+, both equilibrated in urea buffer. After binding, the suspension with the affinity resins was aliquoted in eight portions, four for each resin. After the resins had settled at the bottom of the tubes, the supernatant with unbound protein was removed leaving 100 μL in every tube.
Automated folding screening
The pipetting robot used (Speedy, Zinsser Analytic AG) has four steel pipetting needles and is equipped with a temperature-controlled plate that keeps all solutions at 16°C–18°C. Protein folding was carried out in a rack containing 2-mL tubes for conditions 1–22 (see Table 1) and 1.5-mL tubes for folding of p22 dynactin that was immobilized on metal chelate resins (conditions 23–30, Table 1). All buffer components for rapid dilution screening were pipetted together from stock solutions. For protein addition steps, the rack was transferred onto a stirrer to ensure effective mixing. Screening conditions 1–22 had a final volume of 1 mL. For conditions 1–20, 20-μL aliquots of denatured protein were added in four steps, divided by 150-min incubation time. In the case of conditions 21 and 22 (CTAB/cyclodextrin system), protein was added in one step to 680 μL CTAB-containing buffer and incubated for 150 min, followed by addition of 300 μL β-cyclodextrin and a further 10-h incubation; 350 μL aliquots were submitted to dialysis and subsequent fluorescence spectroscopy and 400 μL were applied to HIC (see below).
For conditions 23–30, denatured protein bound to affinity beads (prepared as described above) was washed with 1.3 mL urea buffer (see above). All conditions, except for 24 and 26, were washed three times in 1.3 mL folding buffers and incubated for 10 h to induce folding. For conditions 24 and 26, the urea concentration was stepwise decreased by dilution with folding buffer (see Table 1, 15-min intervals) followed by two washes and a 9-h incubation. In summary, all samples were washed five times in total, including a final transfer into the same buffer (20 mM Tris-HCl, pH 7.7, 30 mM NaCl, 2 mM β-mercaptoethanol). Finally, protein was eluted with 400 μL of 20 mM Tris-HCl, pH 7.7 150 mM NaCl, 2 mM β-mercaptoethanol, 500 mM imidazole. The obtained samples were analyzed by fluorescence spectroscopy before application to the analytical HIC column (see below).
Tryptophan fluorescence spectroscopy
Three hundred fifty microliters of samples of conditions 1–22 of the folding screen were dialyzed for 2 days against 20 mM Tris-HCl, pH 7.7, 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT, using a membrane with a 4000–6000-Dalton cutoff (Zellu Trans, Roth). Samples obtained from conditions 23–30 were not dialyzed. Three hundred twenty microliters of all samples were filtrated through a 96-well 0.22-μm PVDF membrane (Millipore) and collected in a black 96-well plate (Corning) that has a UV-transparent bottom. A280 and A340 were measured before fluorescence emission spectra were taken between 285 nm and 420 nm upon excitation at 280 nm (Cary Eclipse, Varian Instruments). The reference spectrum was subtracted from the data.
Hydrophobic interaction chromatography (HIC)
To 400 μL aliquots from the folding screen (see above) 3.8 M ammonium sulfate was added under vigorous stirring to achieve a final ammonium sulfate concentration of 2.0 M. Samples were centrifuged (30 min, 25,000 × g, 8°C) and applied to a POROS HP2 column (0.83 mL bed volume, Applied Biosystems), equilibrated in 20 mM Tris-HCl, pH 7.7, 2.0 M ammonium sulfate. Chromatography was performed at 8°C (Vision workstation, Applied Biosystems). The column was washed with equilibration buffer and a gradient to 0 M ammonium sulfate was performed over 18, 14, or 10 column volumes for CAB, MDH, and p22 dynactin, respectively. After each run, the column was washed with 5 M GdmHCl to regenerate the column. The flow rate was 3.5 mL/min; each run took approximately 15 min. The relatively low absorption coefficient at 280 nm (ε = 10,264 (M cm)−1) of the 36.6-kD monomeric MDH prompted us to use the absorbance at 220 nm to detect elution peaks from HIC for this protein. All shown chromatograms were baseline corrected.
Determination of CAB and MDH activity
CAB activities were measured using the pNPAc esterase assay (Rozema and Gellman 1996). Fifty microliters of dialyzed samples from the refolding screen were pipetted into 400 μL 20 mM Tris-HCl, pH 7.7. After addition of 45 μL of 52 mM pNPAc in dry acetonitril and mixing, the formation of the hydrolysis product, p-nitrophenolate, was monitored by measuring the linear increase in absorbance at 400 nm from 30 to 60 sec (background hydrolysis was subtracted). MDH activities were assayed as described before (Hutchinson et al. 1994). The measured activities for both CAB and MDH were related to the activity of a control sample with native enzymes. Absorbance changes were measured using a spectrophotometer (Cary 50 Scan, Varian instruments).
Large-scale purification of p22 dynactin
Inclusion bodies of p22 dynactin from 4-L cultures were prepared and purified as described above. Fifteen milliliters of pure denatured protein (5 mg/mL) in 20 mM Tris-HCl, pH 7.5, 8 M urea, 30 mM NaCl, 15 mM DTT, 1 mM EDTA, 300 mM imidazole was diluted into 510 mL of 20 mM Tris-HCl, 50 mM NaCl, 0.2 mM CaCl2/MgCl2, 0.57 mM CTAB, 2 mM β-mercaptoethanol under vigorous stirring. After 2-h incubation, 225 mL of 15.7 mM β-cyclodextrin was added under vigorous stirring to induce folding at 18°C. After 1 h, 7.5 mg TEV-protease was added, followed by an overnight incubation to cut off the His-tag and to ensure that the folding reaction is completed. After centrifugation to remove precipitates, the folded protein was applied to anion exchange chromatography (POROS HQ, Applied Biosystems) where p22 dynactin did not bind. Subsequently, the p22 dynactin/TEV protease mixture was applied to NiNTA-superose (Qiagen) to remove the TEV-protease. To the flowthrough containing p22 dynactin, 3.8 M ammonium sulfate was added under vigorous stirring to achieve a final ammonium sulfate concentration of 2.2 M. The resulting 2.2 L were applied onto a POROS HP2 column (7.8-mL bed volume) and purified as described above. The protein eluate from the ammonium sulfate gradient was applied to a Superose 12 16/50 column (Pharmacia), equilibrated in 20 mM Tris-HCl, pH 7.7, 50 mM NaCl. Eluted protein fractions that correspond to monomeric p22 dynactin (determined using standard molecular weight markers) were collected. A yield of 25 mg of p22 dynactin was obtained in 25 mL, representing 33% of the urea-denatured protein subjected to the purification procedure. An aliquot of the gel filtration eluate was directly analyzed with CD spectroscopy. The protein was concentrated to a final concentration of 24.7 mg/mL by ultrafiltration (Biomax centrifuge tubes, Millipore). It was examined for occurrence of aggregation, by DLS using the Laser SpectroScatter201 (RiNA GmbH, Germany). The purity of the sample was assessed by SDS-PAGE (Laemmli 1970), using Coomassie staining.
UV CD spectroscopy
A Jasco 715 CD spectropolarimeter, calibrated with camphor sulfonic acid, was used for the CD experiment. The concentration of p22 dynactin in 20 mM Tris/Cl, 50 mM NaCl, pH 7.7 was 1.03 mg/mL. Data were averaged at 10°C over 25 scans accumulated at 0.5-nm intervals with 50-nm/min scan speed and 1-sec response time. In the far UV, overlapping spectra were measured in 1 mm (250–207 nm) and 0.1 ± 0.005 mm (250–186 nm) path-length cells (Hellma, Müllheim, Germany). Spectra were baseline corrected, and the overlapping part of the spectra was used to correct for variations in the path length of the 0.1-mm cell. The spectrum in the near-UV region (350–250 nm) was measured in a 4-mm path-length cell calibrated by absorption spectroscopy.