Folding screening assayed by proteolysis: application to various cystine deletion mutants of vascular endothelial growth factor

Christoph Heiring and Yves A. Muller1,

Forschungsgruppe Kristallographie, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13092 Berlin, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The production of recombinant proteins in Escherichia coli often leads to the formation of inclusion bodies. Although this has a number of advantages, a major disadvantage is the need to develop folding protocols for the renaturing of the proteins. However, the systematic screening of folding conditions is often hampered by the lack of convenient assays to detect correctly folded proteins. To address this problem we present a simple protocol, which combines folding screens and limited proteolysis to rapidly assess and optimize folding conditions. The efficacy of this method, termed FSAP (folding screening assayed by proteolysis), is demonstrated by the large-scale folding, purification and crystallization of various cystine deletion mutants of the cystine knot family member: vascular endothelial growth factor (VEGF). These mutants are particularly difficult to fold as the cystine knot is believed to make major contributions to the stability of the protein and this family of proteins lacks extensive hydrophobic core regions.

Keywords: cystine knot growth factor family/inclusion bodies/proteolysis/renaturing/screening


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The heterologous production of proteins in the prokaryotic host Escherichia coli is a widely used technique both in research and in industrial manufacturing. However, often the proteins are not released from the ribosomes in a soluble and native-like conformation but rather form high molecular weight aggregates that accumulate in inclusion bodies. These inclusion bodies necessitate the development of protocols by which proteins can be solubilized and folded into their biologically active form (Rudolph and Lilie, 1996Go; Lilie et al., 1998Go). Although such folding procedures may prove difficult to develop, inclusion bodies do have advantages compared with cytoplasmatic or periplasmatic accumulation of proteins. For example, a protein, which can be expressed in high yields using strong promoter systems and accumulates in inclusion bodies, is less prone to proteolytic degradation and potential host cell toxicity is minimized. In addition, the protein can be easily separated from E.coli proteins using simple washing and centrifugation steps, rendering the use of affinity tags unnecessary. However, all these advantages become irrelevant in cases where no suitable folding conditions can be found.

In recent years, significant progress was achieved in establishing general guidelines for protein folding (Rudolph et al., 1997Go) which eventually led to the development of factorial folding screens (Chen and Gouaux, 1997Go; Armstrong et al., 1999Go). Unfortunately, the application of these screens is hampered as it is difficult to assess whether the folding was successful or not in the absence of protein-specific enzymatic or biological assays. Our method of folding screening assayed by proteolysis (FSAP) permits the rapid assessment of the folding success directly out of the folding buffers without any further purification. The method, which uses as little as 10 µg of protein in each experiment, relies on the limited proteolysis analysis of the folding products.

To probe the effectiveness of this method, the folding of all four possible cystine deletion mutants of the angiogenic hormone vascular endothelial growth factor (VEGF) was attempted. VEGF is a homodimeric protein and belongs to the cystine knot family of growth factors (McDonald and Hendrickson, 1993Go; Muller et al., 1997Go). Because of the absence of a typical hydrophobic core region, the cystine knot is believed to be a major determinant for the stability of these growth factors. Thus, finding folding protocols for these deletion mutants could be particularly challenging. Nevertheless, our screening method enabled us to derive folding conditions for three out of the four possible cystine deletion mutants of VEGF (Figure 1Go). The developed folding protocols differ from the wild-type VEGF protocol (Christinger et al., 1996Go) and can be readily scaled up to produce proteins in milligram quantities.



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Fig. 1. Sketch of the dimeric growth factor VEGF with the cystine knot emphasized. The two monomers forming the disulfide-bridged homodimer are displayed in light and dark grey. The mutated cysteine residues are labelled. Figure generated with MOLSCRIPT (Kraulis, 1991Go) and RASTER3D (Merritt and Murphy, 1994Go).

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein production

The gene encoding for residues 14–108 of human VEGF was a generous gift from Jerini Bio Tools (Berlin, Germany) and was inserted into a pET-3d vector (Novagen, Madison, WI). Site-directed mutagenesis experiments were performed using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA) following the instructions of the manufacturer. Inclusion bodies of wild-type VEGF were produced in BL21(DE3) cells (Novagen) while higher yields of inclusion bodies were obtained for all mutants by transforming the respective expression plasmid into B834(DE3)pLysS cells (Novagen) co-transformed with the pREP4 vector containing the genes encoding for GroES/GroEL (Caspers et al., 1994Go). However, no specific selection for the pREP4 vector containing host cells was included in subsequent steps. For each construct, 6 l of LB medium (supplemented with 100 µM ampicillin) were inoculated in shake flasks with an overnight culture. After 3 h of growth at 37°C up to an OD600 of ~0.6, overproduction was induced by addition of 1 mM IPTG and the cells were incubated at 37°C for an additional 4 h. Cells were harvested by centrifugation (20 min, 5000 g, 4°C), resuspended in 40 ml of 20 mM Tris–HCl buffer, pH 7.5, supplemented with 5 mM EDTA and disrupted in a French press (2x1000 bar). The crude inclusion bodies were isolated by centrifugation (12 000 g, 4°C, 60 min) and washed three times with the buffer from above and centrifuged each time (12 000 g, 4°C, 30 min). The pellets of the mutants were dissolved in 20 ml of denaturing buffer (20 mM Tris–HCl, pH 8.0, 6 M guanidine–HCl, 5 mM EDTA, 4 mM DTT), stirred for 2 h at room temperature and centrifuged (46 000 g, 4°C, 30 min) and the supernatant was stored at –20°C. In the case of wild-type VEGF and as previously described, 7.5 M urea was used, instead, to dissolve the pellet (Christinger et al., 1996Go).

Folding screen and limited proteolysis assay

The folding screen consisted of 16 solutions closely related to those proposed by Chen and Gouaux (screen conditions 1–16, Table IGo) (Chen and Gouaux, 1997Go; Armstrong et al., 1999Go) together with eight variations of a folding condition previously reported for wild-type VEGF (Christinger et al., 1996Go) (screen conditions 16–24). A 1 ml volume of the dissolved inclusion bodies was diluted ~10-fold in denaturing buffer (see above), leading to a final total protein concentration of 1 mg/ml and as estimated by measuring the UV absorbance at 280 nm. Aliquots of 10 µl of this solution were added to 990 µl of screen solution in an Eppendorf tube, vigorously shaken for 2 min and incubated overnight at 4°C.


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Table I. Composition of the folding solutions
 
The following day, all 24 probes were dialysed for 6 h against 100 ml of the proteolysis buffer (20 mM Tris–HCl, pH 8.0) and each sample was concentrated to 100 µl using Millipore (Bedford, MA) Ultrafree centrifugal devices (molecular weight cut-off 3.5 kDa). After transferring the samples into fresh tubes, 2 µl of a subtilisin solution (0.05 mg/ml) (Boehringer Mannheim, Mannheim, Germany) were added to each probe, resulting in a protease to substrate ratio of 1:100. The proteolysis was aborted after 100 min by adding 10 µl of 10 mM PMSF solution. All samples were vacuum dried, dissolved in 10 µl of standard loading buffer and analysed by standard SDS–PAGE and silver staining.

Wild-type VEGF and soluble misfolded mutant Cys26Ala–Cys68Ala

As a reference, wild-type VEGF was folded as previously described (Christinger et al., 1996Go). Briefly, inclusion bodies of wild-type VEGF were dissolved in 7.5 M urea and diluted about 10-fold into a solution containing 20 mM Tris–HCl, pH 8.4, and 7 µM CuCl2. The protein solution with a total protein concentration of ~1 mg/ml was stirred overnight at room temperature and wild-type VEGF purified subsequently by ion-exchange, hydrophobic interaction and size exclusion chromatography. Attempts to apply the identical folding procedure to the mutant Cys26Ala–Cys68Ala generated a soluble protein fraction, which could not be further purified. During ion-exchange chromatography, the mutant protein eluted at unusually high salt concentrations and over a broad segment of the gradient. Attempts to elute the protein from a hydrophobic interaction column failed completely. Hence we deduced that the protein is misfolded and used this sample of Cys26Ala–Cys68Ala in the following as a reference.

Sensitivity of the proteolytic assay

In order to test how well the proteolytic assay can discriminate between native-like and misfolded VEGF, we prepared three different test mixtures with ratios of 0, 5 and 100% of correctly folded wild-type VEGF to misfolded mutant. To each solution, 1.0 µg of subtilisin was added to 100 µg of total protein in 100 µl of 20 mM Tris–HCl-buffer, pH 8.0. Aliquots of 10 µl were taken at various time intervals and immediately boiled after addition of 5 µl of SDS–sample buffer. Finally, the samples were analysed on SDS–PAGE gels.

Protein concentration and folding efficiency

The folding efficiency was probed for three different concentrations of the mutant Cys61Ala–Cys104Ala by diluting 10 µl of solubilized inclusion bodies with folding buffer (condition 10, Table IGo). Three different protein concentrations were generated, namely 0.01, 0.1 and 1.0 mg/ml. Whereas in the first two cases, the final guanidine–HCl concentration was 0.06 M, for technical reasons and unintentionally the concentration was 0.6 M in the last case. Following folding, equal amounts of protein (10 µg) were removed, the buffer was exchanged and the samples were concentrated to 100 µl prior to the proteolytic digestion and analysis by SDS–PAGE.

Milligram-scale folding, purification and crystallization of the mutants

Prior to the milligram-scale folding, the solubilized inclusion bodies were purified by size exclusion chromatography under denaturing conditions (20 mM Tris–HCl, pH 7.5, 6 M guanidine–HCl, 4 mM DTT) on a Sephacryl S-200 column (Pharmacia, Uppsala, Sweden). A 20 ml volume of the pooled fractions containing ~1 mg/ml protein was diluted into 2 l of folding buffer [50 mM Tris–HCl, pH 8.2, 10.5 mM NaCl, 0.44 mM KCl, 1 mM EDTA, 0.3 mM lauryl maltoside, 440 mM sucrose, 1 mM reduced glutathione (GSH), 0.1 mM oxidized glutathione (GSSG)] and stirred overnight at 4°C. The next day, the solution was dialysed twice for 6 h against 35 l of 20 mM Tris–HCl buffer, pH 8.0. After filtration, the protein was concentrated on a Q-Sepharose column (Pharmacia) and eluted with a 0–1 M NaCl gradient in 20 mM Tris–HCl, pH 8.0. The pooled fractions were concentrated to a volume of 1 ml and further purified by size exclusion chromatography using a Superdex 75 column (Pharmacia).

Crystals of the three mutants were grown by vapor diffusion at 20°C using the hanging drop method. A 1 µl volume of protein solution (12 mg/ml protein, 20 mM Tris–HCl, pH 8.0, 100 mM NaCl) was mixed with 1 µl of reservoir solution and equilibrated against 700 µl of the latter. In the case of the mutants Cys51Ala–Cys60Ala and Cys61Ala–Cys104Ala, the reservoir consisted of 28% (w/v) PEG 4000, 12% (v/v) 2-propanol, 100 mM Na-citrate, pH 5.6, and, in the case of mutant Cys57Ala–Cys102Ala, of 2.0 M Na-formate and 0.1 M Na-acetate, pH 4.6.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Folding screening and subsequent detection of native-like protein by limited proteolysis

Folding conditions for three cystine deficient double mutants of VEGF, in which disulfide-bridged cysteines were replaced by alanine pairs, were identified in a slightly modified version of a well-established folding screen (Chen and Gouaux, 1997Go). For each mutant, aliquots containing 10 µg of solubilized inclusion bodies were diluted to 0.01 mg/ml under 24 different folding conditions (Table IGo), incubated overnight, dialysed and concentrated 10-fold. Successful folding conditions could be identified based on the ability of the products to withstand proteolytic degradation by the protease subtilisin over a 100 min incubation period and at a protease to protein ratio of 1:100. Although as little as 10 µg of total protein were used in each folding experiment, the successful conditions could be readily identified on silver-stained SDS–PAGE gels (Figure 2Go).



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Fig. 2. Folding conditions taken from Table IGo screened by limited proteolysis and analysed by SDS–PAGE. Whereas several promising conditions could be identified for the mutant Cys51Ala–Cys60Ala, only two potential conditions (conditions 8 and 10) could be identified for the mutant Cys61Ala–Cys104Ala. Conditions 8 and 10 were also observed for the mutant Cys57Ala–Cys102Ala, whereas no condition could be identified for the mutant Cys26Ala–Cys68Ala (data not shown). Molecular weight markers are labelled with R.

 
In the case of the mutant Cys51Ala–Cys60Ala, five major conditions (conditions 4, 8, 10, 16 and 24, Table IGo, Figure 2Go) could be identified which share common properties. Either 0.44 M sucrose alone or in combination with 0.55 M arginine successfully enhanced folding yields. Also, all conditions contained either of the detergents ß-lauryl maltoside or CHAPS, whereas no single preference was detectable for pH, sodium chloride, potassium chloride, MgCl2, CaCl2, DTT and various GSSG/GSH ratios (Table IGo). Only one major (condition 10) and one minor folding condition (condition 8) were found for the mutants Cys61Ala–Cys104Ala and Cys57Ala–Cys102Ala, whereas mutant Cys26Ala–Cys68Ala did not fold under any of the reported screening conditions. Our assay thus immediately revealed that detergents and substances such as sucrose and arginine have beneficial effects on the folding of VEGF and that further explorations of these conditions might help to derive folding conditions for the mutant Cys26Ala–Cys68Ala.

Selectivity and sensitivity of the proteolytic assay

Even in well-studied systems, protein folding yields are never particularly high. Therefore, we tried to quantify the percentage of properly folded material necessary in order to be detectable by our proteolytic assay. In order to mimic different folding yields, we mixed properly folded wild-type VEGF with different amounts of a misfolded soluble sample of the mutant Cys57Ala–Cys102Ala (see experimental protocols) and subjected the different mixtures to the proteolytic cleavage assay (Figure 3Go). The time course of the degradation shows that for the misfolded sample the protein is rapidly degraded, whereas for the wild-type VEGF even after 150 min no detectable degradation occurs (Figure 3Go). When investigating different ratios of folded to misfolded protein for VEGF, the proteolytic digestion appears to be extremely discriminative as ratios as low as 5% can be detected (Figure 3Go).



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Fig. 3. Minimal folding yields required for the identification of a potential folding condition. Time courses of the proteolytic degradation starting from 100, 95 and 0% misfolded protein. The results suggest that it is possible to identify folding conditions, even if these yield as little as 5% of properly folded material. The molecular weight markers and wild-type VEGF are labelled with M and R, respectively.

 
Optimal protein concentration during folding

Assuming that the intensity of the protein staining on the SDS gel after proteolysis correlates with the amount of protein that has been successfully folded, we performed the folding of the Cys61Ala–Cys104Ala mutant with screen solution 10 at various protein concentrations, namely at 0.01, 0.1 and 1.0 mg/ml. The denaturing chaotropic agent was diluted 1:100 in the first two experimental conditions and 1:10 in the last condition (60 versus 600 mM guanidine–HCl). Protein concentrations lower than 0.01 mg/ml are not really practicable considering the practical problems arising from the handling of large volumes of folding buffer required for the scale-up of the protocol.

After incubation overnight, 10 µg of total protein were subjected to proteolytic cleavage and analysed by SDS–PAGE, revealing that the folding yield is indeed increased in highly diluted solutions (Figure 4Go). The 0.01 mg/ml setup produces a band which is comparable to the previous experiments, whereas the two other lanes show a significantly weaker band. It is well known that folding competes with aggregation and that dilution promotes the intramolecular folding process versus the intermolecular aggregation (Lilie et al., 1998Go). Therefore, these experiments may help to determine the optimal concentration in which to fold a particular protein.



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Fig. 4. Influence of the protein concentration on the folding yields. Equal amounts of protein obtained from three different folding experiments in which the protein concentration in the folding buffer was varied were subjected to limited proteolysis. Analysis of the products by SDS–PAGE shows that higher folding yields are obtained when low protein concentrations are used during folding.

 
Large-scale folding and conformational homogeneity of the products

To confirm whether the folding conditions obtained for the three mutants could be scaled to large volumes and yield homogeneous native-like protein, we generated several milligrams of each of the three mutants. For each mutant, 20 mg of protein, prepurified and solubilized in 6 M guanidine–HCl buffer, were rapidly diluted into 2 l of folding buffer (condition 10, Table IGo). After folding and dialysis, the protein was concentrated on an ion-exchange column and further purified by gel filtration. Each mutant eluted in a sharp peak with almost no further contamination (Figure 5Go). The final yields for the mutants Cys61Ala–Cys104Ala, Cys51Ala–Cys60Ala and Cys57Ala–Cys102Ala were 5, 3 and 1.5 mg, respectively. These yields reflect surprisingly well the relative yields observed in the screening assay. In fact, the absolute yields appear to be a factor of two higher than expected and this might be the result of the pre-purification of the mutants prior to folding.



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Fig. 5. Size exclusion chromatography elution profile of the mutant Cys61Ala–Cys104Ala. A single crystal of the same mutant is shown in the inset.

 
Crystallization screens yielded crystals for all three mutants within 2 days. The crystals are of high quality and show similar morphological and crystallographic properties as crystals of wild-type VEGF (data not shown). Both the chromatographic elution properties and the crystal quality of the mutant proteins are strong indicators for the proteins' conformational homogeneity.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The idea that limited proteolytic digestion might provide a general method to monitor protein folding has been proposed before (Rudolph et al., 1997Go) and in numerous studies partial proteolytic cleavage has been used to unveil protein folding pathways (Ooi et al., 1963Go; Schmid and Blaschek, 1984Go; Teschner and Rudolph, 1989Go). However, to the best of our knowledge, this is the first time that folding conditions for novel proteins have been rapidly ascertained by this method. The potential of this method for general applicability is greatly expanded when combined with recently developed fractional factorial protein folding screens (Chen and Gouaux, 1997Go; Armstrong et al., 1999Go).

Applying this method, we were able to derive folding conditions for three of four possible mutants of VEGF in which individual disulfide bridges have been removed. Using as little as 10 µg of protein for each folding experiment allowed the assessment of the folding success and folding yields as low as 5% were detectable by the limited proteolysis of the products. Furthermore, this method provides a simple protocol to study the effect of dilution on folding yields. The fact that the screening of folding conditions was performed on crude extracts from washed and solubilized inclusion bodies highlights the robustness of this method.

VEGF is possibly especially well suited for this kind of analysis as we observe an extreme discrimination between misfolded and native-like protein in the partial proteolysis experiments. However, the fact that limited proteolysis is commonly used in structural and functional studies for the detection and delineation of globular folding units (Porter, 1973Go; Nakagawa et al., 1997Go; Schwartz et al., 1999Go) suggests that such discriminative conditions can be established for most proteins. Small amounts of properly folded protein, possibly from transient expression in mammalian cells, should help to find such discriminative conditions. If folded protein is unavailable, the wealth of information available on limited proteolysis allows general proteolysis protocols to be used.

Any protease could be used with the FSAP method as a number of proteases such as thermolysin, chymotrypsin, trypsin, subtilisin and SV8-protease are often used in limited proteolysis experiments. However, the problem of discriminating between misfolded and native-like protein may differ slightly from dissecting a folded protein chain into its functional globular domains. In the latter case, the targeted cleavage sites are anticipated to be part of flexible, predominantly hydrophilic loop segments, which connect independent folding units. However, in misfolded proteins hydrophobic segments might be accumulated on the surface of the protein when compared with the native-like protein. Thus subtilisin, which cleaves after hydrophilic residues and which was used here, might not be the best choice and it might be worth investigating alternative proteases with preferences for hydrophobic stretches.

A major advantage of our folding assay is the fact that it provides for a rapid general screening method, which easily distinguishes between misfolded and native-like protein without the need for the prior establishment of a specific biological assay for protein function. In contrast to biophysical methods, such as dynamic light scattering and circular dichroism measurements, commonly used to confirm the folding state of proteins, the FSAP method does not depend on the conformational uniformity of the solution. Rather, it can be performed on mixtures of native-like and misfolded proteins. In addition, the method can be easily automated and thus the folding screens and the proteolytic assays varied extensively. For example, it can be easily envisaged to use multi-well plates for the folding together with serial dialysis devices as well as the on-line detection of the products by capillary electrophoresis or mass spectrometry.

Because of this potential for high automation, the FSAP method might be of particular interest in structural genomics projects. Here the systematic production of proteins with only a limited knowledge available on their biological function and biophysical properties is a prerequisite.


    Notes
 
1 To whom correspondence should be addressed. E-mail: yam{at}mdc-berlin.de Back


    Acknowledgments
 
This work would not have been possible without the generous support of Udo Heinemann. We thank Gisela Sklenar for advice and Udo Heinemann and Heinz Welfle for numerous discussions and together with Celia Schiffer for critical reading of the manuscript. This work was supported in part by the Deutsche Forschungsgemeinschaft, SFB449-B8.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received November 16, 2000; revised January 10, 2001; accepted January 10, 2001.