A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein

Yves Nominé1, Tutik Ristriani2, Cécile Laurent2, Jean-François Lefèvre1, Étienne Weiss2 and Gilles Travé1,3

1 Laboratoire de RMN, UPR 9003 du CNRS and 2 Laboratoire d'Immunotechnologie, UPRES 1329, École Supérieure de Biotechnologie de Strasbourg, 67400 Illkirch, France


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recombinant production of HPV oncoprotein E6 is notoriously difficult. The unfused sequence is produced in inclusion bodies. By contrast, fusions of E6 to the C-terminus of carrier proteins such as maltose-binding protein or gluthatione-S-transferase are produced soluble. However, it has not yet been possible to purify E6 protein from such fusion constructs. Here, we show that this was due to the biophysical heterogeneity of the fusion preparations. We find that soluble MBP-E6 preparations contain two subpopulations. A major fraction is aggregated and contains exclusively misfolded E6 moieties (`soluble inclusion bodies'). A minor fraction is monodisperse and contains the properly folded E6 moieties. Using monodispersity as a screening criterion, we optimized the expression conditions, the purification process and the sequence of E6, finally obtaining stable monodisperse MBP-E6 preparations. In contrast to aggregated MBP-E6, these preparations yielded fully soluble E6 after proteolytic removal of MBP. Once purified, these E6 proteins are stable, folded and biologically active. The first biophysical measurements on pure E6 were performed. This work shows that solubility is not a sufficient criterion to check that the passenger protein in a fusion construct is properly folded and active. By contrast, monodispersity appears as a better quality criterion. The monodispersity-based strategy presented here constitutes a general method to prepare fusion proteins with optimized folding and biological activity.

Keywords: fusion protein/HPV E6 oncoprotein/monodispersity/proper folding/quality optimization


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In recent years, bacterial overproduction of heterologous polypeptides in bacteria has become a routine matter (Banyex, 1999Go). However, a high protein synthesis rate is necessary but not sufficient for the efficient production of biologically active proteins. The overexpressed polypeptide chain has to fold into the correct native three-dimensional structure. Frequently, only a fraction of the recombinant polypeptides achieves proper folding, the rest being produced misfolded and accumulating in inclusion bodies (Georgiou and Valax, 1996Go; Banyex, 1999Go). In such cases, it is important to screen for expression and purification conditions favouring the folded form. This requires a standard measurement allowing one to quantify the percentage of folded polypeptides obtained under each condition tested. This measurement can be based on biological activity, but this implies that a new assay has to be developed for every new protein studied. Moreover, in many cases, the protein or protein domain studied does not display any known activity at all. A classical alternative to biological assays consists in evaluating the percentage of soluble protein produced. Provided that the protein of interest is cloned as a genuine unfused sequence, solubility may be regarded as a universal check for proper folding, since misfolded proteins generally precipitate. However, recombinant proteins or domains are very often fused to carrier proteins such as maltose-binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (Trx) (LaVallie and McCoy, 1995Go). Fusion proteins have become crucial tools for recombinant protein production. They are especially useful for affinity purification of recombinant proteins and detection of their specific interactions with biological ligands, including other proteins or peptides. Many workers in the field tend to extend to fusion proteins the paradigm that solubility is a guarantee of proper folding and biological activity. However, several reports have contradicted this view, by showing that soluble preparations of a fusion protein may display low biological activity or no activity at all, as compared with the native protein from natural sources (Louis et al., 1991Go; Saavedra-Alanis et al., 1994Go; Lorenzo et al., 1997Go; Sachdev and Chirgwin, 1998Go, 1999Go). These findings stressed the need for a universal criterion, other than solubility, which would allow one to evaluate the quality of fusion proteins.

E6 is an oncoprotein produced by the `high-risk' human papillomaviruses (HPVs) responsible for 95% of cervical cancers (Scheffner et al., 1990Go). More than 100 E6-encoding DNA sequences have been published since 1985 (Chan et al., 1995Go). Despite this fact, there is little information about the molecular properties of the protein. The 3D structure is not known and even easy biophysical measurements concerning the optical properties of the protein (UV absorption, UV fluorescence, circular dichroism) are missing. This lack of information is certainly related to the absence of a proper protocol allowing one to purify stable samples of soluble and biologically active recombinant E6. The genuine E6 sequence, either unfused or His-tagged, is mainly produced as inclusion bodies (Imai et al., 1989Go). In the contrast, fusions of E6 to the C-terminus of either GST (GST-E6) or (MBP-E6) are overexpressed in a soluble form in Escherichia coli and can be purified using the appropriate single-step affinity procedures (Lechner and Laimins, 1994Go; Kukimoto et al., 1998Go). However, proteolytic removal of GST (or MBP) from the GST-E6 (MBP-E6) fusions leads to rapid precipitation of the E6 protein (Lechner and Laimins, 1994Go).

By using independent biochemical, biophysical and immunological methods, we have recently found that MBP-E6 preparations contain multimeric aggregates composed of folded, active MBP moieties fused to agglomerated misfolded E6 chains (Nominé et al., 2001Go). These soluble aggregates have been named `soluble inclusion bodies' by analogy with the solid aggregates which are normally formed by unfused misfolded proteins overexpressed in bacteria. In contrast to the solid inclusion bodies, these soluble inclusion bodies co-purify with the proper form of the fusion protein on affinity resins, so that they contaminate the final `pure' preparations. Therefore, for fusion proteins, solubility does not always correlate with proper folding of the passenger protein. Monodispersity appears to be more appropriate than solubility for quality checking of fusion proteins. Here, we used this concept to solve the problem of overproduction of soluble, folded and biologically active recombinant E6 protein. We performed aggregation measurements to screen the conditions favouring monodispersity of MBP-E6 preparations. In a stepwise process, we successively optimized the expression conditions, the purification protocol and the amino acid sequence of the E6 moiety, finally obtaining stable monodisperse MBP-E6 preparations. In contrast to the initial aggregated MBP-E6 preparations, these optimized preparations yielded fully soluble E6 proteins after proteolytic removal of the MBP moiety. We purified these E6 proteins to homogeneity and showed that they are stable, folded and biologically active.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression and purification of MBP-E6 fusions

The genes encoding the 158 residues of wild-type (wt) or mutated HPV16 E6 were cloned into plasmid pMAL-c2 vector (New England Biolabs), generating a fusion with the malE gene which encodes MBP. A thrombin restriction site was introduced between MBP and E6 to replace the factor Xa restriction site which was initially present. We developed a standard protocol for expression and rapid purification of small-scale quantities of MBP-fusions. Initially, the MBP-E6 fusions were expressed for 3 h at 37°C in the XL1 Blue E.coli strain. After optimization MBP-E6 fusions were expressed overnight at 27°C in XL1blue E.coli carrying a co-expression vector producing the GroEl chaperone (kindly provided by Dr J.Chatellier, MRC, Cambridge, UK). Cells from a 10 ml culture were harvested by centrifugation and resuspended in 1 ml of buffer containing 1 µg/ml DNase I and 1 µg/ml RNase I. Anti-protease cocktail (EDTA-free) (Boehringer Manheim) was added at the concentration recommended by manufacturer. The initial buffer was buffer A (Tris 50 mM, NaCl 200 mM, pH7.5, ZnSO4 200 µM and was changed to buffer B [phosphate 50 mM, NaCl 200 mM, dithiothreitol (DTT) pH 7.4] after optimization. For the MBP-E6 6C/6S mutant, we used buffer B without DTT. Cells were broken by sonication on ice, then centrifuged at 30 000 r.p.m. (80 000 g) for 15 min at 4°C. The supernatant was filtered and incubated for 30 min with 150 µl of amylose resin (New England Biolabs) equilibrated with same buffer. The mixture was centrifuged for 5 min at 600 r.p.m. (75 g). The liquid was discarded and the resin was washed three times for 10 min with 14 ml of buffer. The resin was then incubated for 5 min with 100 µl of buffer containing 20 mM maltose to allow elution of MBP fusions from resin. Complete extraction of total liquid present inside and outside the resin could be achieved by transfering the resin–liquid mixture to a 0.5 ml Eppendorf tube possessing a pinhole and filled with glass-wool. Centrifugation into a 2 ml tube allowed the extraction of the liquid while the dried resin was retained by the glass-wool. The total purification procedure took ~1.5 h and could be applied to 18–24 different samples in parallel. The purified proteins were analysed by SDS–PAGE and subjected to aggregation measurements on a fluorimeter (as described in Determination of the aggregation rate). After optimization, an additional step was included in the purification. The MBP-E6 fusions extracted from amylose resin were incubated with 100 µl of S-Sepharose resin for 1 h in 1 ml of buffer B low-salt (NaCl 50 mM). The liquid was discarded and the resin was washed three times for 30 min with 14 ml of same buffer. The resin was then incubated for 10 min with 100 µl of buffer B high-salt (1200 mM) to allow elution of monomeric MBP-E6 fusions from the resin. Complete extraction of liquid was achieved as described above.

To purify unfused E6 6C/6S, the preparation of monodisperse MBP-E6 6C/6S was scaled up to start from 500 ml of expressed culture; the amylose and S-Sepharose steps were performed on columns. The high-salt eluate of the S-Sepharose column containing monodisperse MBP-E6 6C/6S was first adjusted to 150 mM NaCl, then incubated with thrombin until full separation of E6 from MBP. The digestion product was re-applied on S-Sepharose pre-equilibrated with 20 mM phosphate pH 7.4. After washing, the column was subjected to a gradient of NaCl from 0 to 1 M. Pure monodisperse E6 6C/6S protein was eluted as a single peak at 500 mM NaCl.

Site-directed mutagenesis by the polymerase chain reaction (PCR)

The E6 6C/6S mutant was constructed from the wild-type HPV16 E6 sequence by `overlapping' PCR (Higuchi et al., 1988Go), then subcloned into the pMal-c2 (New England Biolabs) and sequenced using a Thermo Sequenase Cy5.5 dye terminator cycle sequencing kit (Amersham Pharmacia Biotech). Sequences were analysed with Seq4x4 Basecaller and ALFwin sequence analyser.

Construction of the DNA probe for gel-retardation assay

A cruciform based upon the sequence of junction 1 originally described by Duckett et al. (1988) was constructed by hybridizing four oligonucleotides each of 30 nucleotides referred to as b, h, r and x, respectively, one of which was 5'-32P-labelled. These were

b-strand: 5'-TCCGTCCTAGCAAGCCGCTGCTACCGGAAG-3'

h-strand: 5'-CTTCCGGTAGCAGCGAGAGCGGTGGTTGAA-3'

r-strand: 5'-TTCAACCACCGCTCTTCTCAACTGCAGTCT-3'

x-strand: 5'-AGACTGCAGTTGAGAGCTTGCTAGGACGGA-3'

Gel retardation analysis

Purified proteins were mixed with labelled DNA junction in 25 mM Tris–HCl pH 7.5, 100 mM NaCl, 10 mM KCl, 1.5 mM MgCl2, 100 µg/ml genomic DNA and 5% glycerol in a total volume of 10 µl. Samples were then incubated for 15 min on ice and electrophoresed in 6.5% polyacrylamide gels (29:1 acryl:bisacryl) with 45 mM Tris–borate in the presence of 1 mM EDTA, at 10 V/cm at 8°C. Gels were dried on Whatman 3M paper and analysed on a Phosphorimager (Amersham Pharmacia Biotech).

Spectroscopic measurements

Fluorescence and lateral turbidimetry measurements were performed with a SPEX Fluorolog-2 spectrofluorimeter (SPEX Industries, Edison, NJ) equipped with a 450 W Xe lamp, a double-grating excitation monochromator and a single-grating emission monochromator. Data were acquired with a photon counting photomultiplier (linear up to 107 counts/s) with high voltages fixed at 800 V. Slit widths were adjusted to avoid saturation of detectors. Unless stated otherwise, 2 ml of solution were placed in a cuvette maintained at 15°C in a thermostatted cuvette handler. Emission spectra recordings were typically sampled every 0.5 nm. In all experiments, the slit widths were set to 1.8 nm for both excitation and emission. Ultraviolet absorption measurements were performed with a Perkin-Elmer l-2 spectrophotometer. Circular dichroism spectroscopy was performed using a Jobin-Yvon spectropolarimeter.

Determination of the aggregation rate

The samples of aggregated proteins were placed in a fluorimeter at a concentration of ~200 nM and excited by a monochromatic light (280 nm). An emission spectrum was recorded in the range 260–400 nm. A spectrum recorded for buffer alone served as a subtraction baseline for all samples measured. Each experiment was repeated at least three times. We recorded a first maximum intensity at 280 nm (the scattered light) and a second maximum intensity at 340 nm (the fluorescence signal from the protein). From these two values we then calculated an ` aggregation rate' defined as follows:


where I280 and I340 represent the intensity of emitted light at 280 and 340 nm, respectively.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental evaluation of aggregation of MBP-E6 preparations

We have previously observed that MBP-E6 soluble inclusion bodies can be detected at very low concentrations by performing light scattering measurements in a fluorimeter (Nominé et al., 2001Go). The intensity of fluorescence is proportional to the quantity of protein, but the efficiency of fluorescence decreases with aggregation. The intensity of scattered light is also proportional to the quantity of protein and it also increases with the size of the aggregates (Cantor and Schimmel, 1980Go). These effects can be verified by performing a 260–400 nm emission scan on either MBP (monodisperse) or MBP-E6 (aggregated) diluted at the same final concentration and excited by 280 nm radiation (Figure 1AGo). These observations allow one to define a parameter, practically independent of concentration, which quantifies the aggregation state of any preparation of MBP fusion. This parameter, which we will call `aggregation rate', is calculated as follows:



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Fig. 1. Principle of the aggregation rate. (A) Monodisperse MBP and aggregated MBP-E6 were diluted to a final concentration of 200 nM and subjected to 280 nm excitation. An emission scan was recorded in the 26–400 nm range. As indicated by arrows on the MBP-E6 spectrum, the intensity of light scattered at 280 nm increases with aggregation whereas the intensity of fluorescence at 340 nm decreases with aggregation. One can therefore define an `aggregation rate' practically independent of the concentration of the protein measured: Ragg = I280/I340. (B) Aggregation rates obtained for samples of MBP-E6, MBP-SPCI and MBP at a final concentration of 200 nM.

 

where I280 is the light scattered by the solution of MBP fusion as compared with buffer and I340 is the fluorescence of the same solution as compared with buffer. Since the two values are both proportional to the quantity of protein measured, their rate represents a `normalized value' evaluating the aggregation state of the MBP fusion independently of the quantity measured. Therefore, determination of the fusion protein concentration by other methods is, in principle, not necessary. Figure 1BGo shows the aggregation rates obtained for MBP-E6 and MBP preparations measured at the same concentration (200 nM). The MBP-SPCI protein, which consists of a fusion between two monodisperse proteins, was also measured. The difference observed between MBP-E6 and the monodisperse MBP control is unambiguous. By contrast, the rate measured for the MBP-SPCI fusion is almost as low as that obtained for MBP control. This optically determined aggregation rate therefore distinguishes aggregated MBP fusions from monodisperse MBP fusions at very low concentration.

Optimizing monodispersity of MBP-E6 preparations

We designed a strategy aiming to remove the soluble inclusion bodies and to isolate the monodisperse MBP-E6 fraction containing exclusively folded E6 moieties. A rapid mini-preparation protocol (see Materials and methods) was applied to prepare series of MBP-E6 samples, each of them obtained using different conditions of expression or purification. The aggregation rate of each preparation was then systematically evaluated using a spectrofluorimeter as described above. The monodispersity of MBP-E6 preparations was optimized by varying three main conditions. A first set of samples was prepared by varying the expression conditions and keeping the other conditions constant. The expression condition leading to the lowest aggregation rate was used for the next step, in which we tested different purification buffers. The buffer favouring the lowest aggregation rate was then selected and different additional purification steps (mainly chromatography resins other than amylose) were tested. The main progress achieved in our optimization is summarized in Figure 2Go. In the first round of optimization (Figure 2Go, lanes 1 to 2), an 80% decrease in the aggregation rate was obtained by shifting the expression temperature to 27°C and co-expressing a fragment of the GroEl chaperonin. Such conditions are classically found to lower the rate of inclusion body formation.



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Fig. 2. Rational optimization of the monodispersity of MBP-E6 preparations. The plot represents the agregation rates (Ragg) measured before and after each cycle of optimization for MBP-E6 preparations diluted at a concentration of 50–200 nM. Lane 1: agregation rate of MBP-E6 preparations before optimization. Expression was induced for 3 h at 37°C. The protein was purified on amylose using zinc-containing buffer A. Lane 2: optimization of expression conditions. Expression temperature was shifted to 27°C and the bacteria contained an additional plasmid expressing the GroEl chaperone. Lane 3: optimization of purification buffer. Buffer A (Tris–NaCl–zinc, pH 7.5) was changed to buffer B (phosphate–NaCl–DTT, pH 7.4). Lane 4: optimization of purification steps. The amylose eluate was loaded on S-Sepharose resin at low salt (50 mM NaCl). The resin was washed extensively in buffer B, 0 M NaCl. The protein retained on the resin was finally eluted by raising the salt concentration (buffer B with 750 mM NaCl).

 
In the second round of optimization (Figure 2Go, lanes 2 to 3), a 40% decrease in the aggregation rate was further achieved by using a purification buffer devoid of zinc ions and in the presence of the reducing agent DTT. We had initially added zinc ions to the buffer with the purpose of stabilizing the two putative zinc-binding domains of E6. However, zinc is also a strong oxidative agent. This result suggests that wild-type E6 is extremely sensitive to redox conditions in the purification buffer.

In the third round of optimization, we tested the retention of amylose-purified MBP-E6 proteins on several resins, including ion-exchange resins (S-Sepharose, CM-Sepharose, Q-Sepharose and DEAE-Sepharose) as well as a hydrophobic resin (phenyl-Sepharose). The cation-exchange resins Q-Sepharose and DEAE-Sepharose neither retained nor separated the MBP-E6 molecules. In contrast, the MBP-E6 proteins were retained by the hydrophobic resin phenyl-Sepharose. A minor fraction eluted with the addition of ~20% ethylene glycol whereas the majority of MBP-E6 molecules only eluted under more hydrophobic conditions, i.e. when 50% ethylene glycol was added. The minor fraction which eluted at 20% ethylene glycol was found to be less aggregated than the major fraction (data not shown). However, the best retention and separation were obtained with anion-exchange resins. At low ionic strength (50 mM NaCl), the strong anion-exchanger S-Sepharose was found to retain specifically a minor fraction of the MBP-E6 proteins, whereas the major fraction was found in the flow-through. The bound MBP-E6 population eluted at a relatively high ionic strength, above 500 mM NaCl. The aggregation rate of the unbound major protein fraction was comparable to that of the input. In contrast, the rate measured for the high-salt minority eluate was much lower (Figure 2Go, lane 4). A similar result was obtained using the weak anion exchanger CM-Sepharose. In conclusion, our rational monodispersity optimization procedure allowed us to purify a fraction of MBP-E6 protein possessing a remarkably low aggregation rate compared with the initial sample (the final value of the aggregation rate was 25-fold lower than the initial value).

Increasing E6 solubility by site-directed mutagenesis

Our initial amylose resin-purified preparations of MBP-E6 contained a majority of soluble inclusion bodies, i.e. aggregates of misfolded E6 moieties solubilized by folded MBP moieties (Nominé et al., 2001Go). In contrast, the fraction of MBP-E6 resulting from our monodispersity optimization procedure (step 4 in Figure 2Go) was expected to contain a majority of monodisperse proteins. However, we observed that this low-aggregated preparation was prone to re-aggregation under storage. This aggregation was accelerated by oxidative conditions. When the preparation was subjected to linker-specific cleavage, the E6 moieties released by proteolysis were still prone to precipitation and to irreversible retention on purification resins. However, we observed that the rapidity of precipitation could be reduced by raising the concentrations of the reducing agent DTT. These findings were in accordance with our previous observation that the aggregates present in amylose-purified preparations of MBP-E6 were favoured by oxidative conditions (Figure 2Go, lanes 2–3). The HPV16 E6 sequence contains an unusually high proportion of cysteines (14 cysteines out of 158 residues) (Figure 3Go). According to multiple alignments, eight cysteines (underlined) are strictly conserved in all the E6 families. They are considered to be important to the 3D structure by binding two zinc ions (Cole and Danos, 1987Go). In contrast, the six other cysteines are not conserved and are generally substituted by hydrophilic residues. They are probably exposed at the surface of the protein and may therefore participate in the formation of non-specific intermolecular cysteine bridges during the expression and purification processes. We constructed a mutant, named E6 6C/6S, with these six cysteines simultaneously changed to serines (Figure 3Go). To study the purification behaviour of this mutant, we prepared in parallel a sample of wild-type MBP-E6 and a sample of MBP-E6 6C/6S. Both proteins were expressed at 27°C and purified on amylose resin using buffer A. When subjected to thrombin cleavage in solution, the wild-type MBP-E6 generated fully precipitated E6 (Figure 4AGo, left gel). On-resin cleavage of wild-type MBP-E6 generated E6 moieties which all remained irreversibly bound to the resin (Figure 4AGo, right gel). In contrast, in-solution cleavage of the MBP-E6 6C/6S preparation yielded a fraction of soluble E6 6C/6S moieties (Figure 4BGo, left gel). When cleavage was performed on-resin, an equivalent quantity of soluble E6 6C/6S protein was also detected in the extracted liquid fraction (Figure 4BGo, right gel). Therefore, mutating the six non-conserved cysteines of HPV16 E6 generated an increase in solubility of E6 moieties after thrombin cleavage.



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Fig. 3. Amino acid sequences of wild-type E6 and of E6 6C/6S mutant. The full-length HPV16 E6 wild-type sequence is shown. Five regions can be distinguished based on multiple sequence alignements of different HPV types: N-terminal, first zinc-binding domain, interdomain, second zinc-binding domain and C-terminal. Eight cysteines are underlined; they form the zinc-binding domains and are conserved throughout the E6 family. The six additional cysteine residues are not conserved. These six cysteines have been mutated simultaneously into serines (as indicated by an S above the wild-type sequence), to raise the E6 6C/6S mutant.

 


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Fig. 4. Comparison of the properties of E6 and E6 6C/6S moieties after thrombin cleavage. (A) Wild-type MBP-E6. Left gel: in-solution cleavage. MBP-E6 protein was expressed at 37°C and purified on amylose resin in buffer A according to our initial non-optimized conditions. The MBP-E6 proteins were eluted by addition of maltose. The liquid was extracted from the resin, then incubated with thrombin. The digested material was then subjected to ultracentrifugation. The non-cut material (`NC', lane 1), the supernatant (`S', lane 2) and the pellet (`P', lane 3) were analyzed by SDS–PAGE. The MBP protein released by cleavage was mainly found in the supernatant, whereas the E6 protein was only detected in pellet. Right gel: on-resin cleavage. MBP-E6 fusions bound to amylose resin were subjected to thrombin cleavage. After the reaction, the liquid phase (containing the protein eluted from the resin) was extracted and the resin was washed extensively. The non-cut material (`NC', lane 4), the liquid extract (`L', lane 5) and the washed resin beads (`R', lane 6) were analyzed by SDS–PAGE. MBP was found in both the resin and liquid extract, whereas E6 was only found in the resin and was not detected in the liquid. (B) Behaviour of mutated E6 6C/6S. MBP-E6 6C/6S protein was expressed, purified and subjected to thrombin cleavage under exactly the same conditions as used for wild-type MBP-E6 in (A). The bands indicated by arrows (lanes 2 and 5) were not observed for wild-type MBP-E6. They correspond to soluble E6 6C/6S.

 
Monodisperse MBP-E6 6C/6S generates fully soluble E6 6C/6S upon thrombin cleavage

The previous finding prompted us to purify the fraction of MBP-E6 6C/6S which generated soluble E6 6C/6S upon thrombin cleavage. The MBP-E6 6C/6S mutant was expressed and purified following the optimized conditions for the wild-type MBP-E6 construct. As previously observed for wild-type MBP-E6, S-Sepharose separated the aggregated amylose preparation into two protein populations; 80% of the applied protein was not retained, whereas 20% was retained and eluted as a low-aggregated fraction upon addition of 500 mM NaCl. The aggregation rates of the amylose and S-Sepharose eluates of MBP-E6 6C/6S were comparable to those observed for wild-type MBP-E6 under the same conditions (Figure 5AGo). However, in contrast to the wild-type construct, the low-aggregated S-Sepharose extract of MBP-E6 6C/6S released fully soluble E6 6C/6S moities after thrombin cleavage and no protein precipitate was observed (Figure 5BGo). This demonstrated that the soluble E6 6C/6S moieties observed after cleavage of the amylose preparation of MBP-E6 6C/6S (Figure 4Go) were actually produced by the monodisperse MBP-E6 6C/6S population.



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Fig. 5. Purification and cleavage of a low-aggregated MBP-E6 6C/6S fraction. (A) Comparison of aggregation rates of wild-type MBP-E6 and MBP-E6 6C/6S expressed and purified under the same conditions. Wild-type MBP-E6 and MBP-E6 6C/6S were expressed at 27°C and purified in buffer B according to the conditions optimized for wild-type MBP-E6 (see Figure 2Go). Lanes 1–4: aggregation rates measured for amylose eluate of wild-type MBP-E6, S-Sepharose eluate of wild-type MBP-E6, amylose eluate of MBP-E6 6C/6S and S-Sepharose eluate of MBP-E6 6C/6S, respectively. (B) Thrombin cleavage of the S-Sepharose eluate of MBP-E6 6C/6S. The S-Sepharose eluate of MBP-E6 6C/6S was diluted 3-fold to reduce the salt concentration and incubated with thrombin. After incubation, the sample was subjected to ultracentrifugation. The non-cut material (`NC', lane 1), the supernatant (`S', lane 2) and the pellet (`P', lane 3) were analyzed by SDS–PAGE.

 
Complete purification protocol of stable E6 6C/6S

The observations accumulated in the previous steps enabled us to design a complete purification protocol for stable, soluble and monodisperse E6 protein (Figure 6Go). The E6 6C/6S was expressed as an MBP-fusion in bacteria at 27°C in the presence of the mini-chaperon construct. The fused protein was purified on amylose resin, then immediately loaded on an S-Sepharose column; 80% of the protein was not retained, while 20% of the protein was bound and eluted as a sharp peak at 500 mM NaCl. The total amylose preparation and the S-Sepharose eluate have a similar profile on SDS–PAGE but clearly differ in their aggregation characteristics (Figure 5AGo). When low-aggregated S-Sepharose eluate was subjected to thrombin digestion, no precipitation was observed. In contrast, when thrombin digestion was performed on the flow-through, all E6 6C/6S moieties precipitated. The thrombin digest of the low-aggregated fraction was then loaded on a second S-Sepharose column. Pure MBP protein was not retained on the resin, whereas E6 6C/6S was retained and eluted as a sharp peak at 500 mM NaCl. This fraction was monodisperse according to our aggregation measurements. It is extremely stable and could be stored for several months at 4°C, in the absence of DTT, without precipitation.



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Fig. 6. Purification of the stable mutant E6 6C/6S. The MBP-E6 6C/6S mutant protein was expressed in E.coli and purified on amylose resin followed by S-Sepharose chromatography. E6 6C/6S was cleaved from the MBP moiety by thrombin and repurified by S-Sepharose chromatography. The proteins were analysed by SDS–PAGE. Lane 1, total soluble protein of the cell lysate; lane 2, MBP-E6 6C/6S eluted from the amylose column; lane 3, aggregated MBP-E6 6C/6S in the flow-through of the S-Sepharose column; lane 4, monodisperse MBP-E6 6C/6S eluted from the S-Sepharose column; lane 5, soluble fraction of thrombin digest; lane 6, MBP in the flow-through of S-Sepharose; lane 7, S-Sepharose eluate containing pure monodisperse E6 6C/6S protein.

 
Biological activities of pure E6 6C/6S

The purified E6 6C/6S sample was tested for the two E6 activities which can be assayed in vitro. The first activity tested was the stimulation of the degradation of the tumour suppressor p53 in cell extracts. This activity is considered to be responsible for the oncogenic behaviour of E6. In the standard assay, radioactively labelled p53 and E6 were in vitro transcribed in rabbit reticulocyte extracts. In vitro transcribed E6 then promotes the time-dependent degradation of p53 by the ubiquitination system, a cellular machinery for the selective proteolysis of specific target proteins. We reproduced this assay by replacing the mixture containing the in vitro transcribed E6 by mock reticulocyte extracts supplemented with various dilutions of purified E6 6C/6S (Figure 7AGo). Purified E6 6C/6S was active in this test and replaced efficiently in vitro transcribed wild-type E6 at concentrations as low as 10 nM. The second property tested was the DNA-binding activity of E6 (Ristriani et al., 2000Go). The purified E6 6C/6S sample retained this activity, as shown by gel-retardation assay (Figure 7BGo). In conclusion, the E6 6C/6S protein retained the biological activities of wild-type E6, with the advantage that it can be purified in a stable form.



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Fig. 7. Biological activities of the stable mutant E6 6C/6S. (A) Degradation of in vitro translated p53 proteins. p53 was translated in reticulocyte lysate and incubated with either in vitro translated E6 (lane 1) or decreasing quantities (3 µM, 300 nM, 30 nM, 3 nM and 300 pM: lanes 2–6, respectively) of purified mutant E6 6C/6S proteins. t0, Samples were immediately removed from the reaction mixture before incubation at 29°C; t1, samples were removed after 2 h of incubation at 29°C. The results were analysed by SDS–PAGE and phosphorimaging. (B) Binding of E6 6C/6S proteins to four-way DNA junctions. 1 nM of radioactively 32P-labelled junction 1 (Duckett et al., 1988Go) with four arms of 15 base pairs each was incubated with increasing concentrations of E6 6C/6S in the presence of 100 µg/ml genomic DNA and 1 mM magnesium ions. The mixtures were electrophoresed in the presence of 1 mM EDTA, then analysed by phosphorimaging. Lanes 1–8: 1 nM cruciform DNA was incubated with 0, 12.5, 20, 40, 80, 125, 250 and 500 nM E6 6C/6S, respectively.

 
Characterization of the E6 6C/6S protein by optical methods

To check the purity and quality of the E6 6C/6S preparation, this sample was first subjected to UV absorption measurements (Figure 8AGo). Note that the E6 6C/6S spectrum has a particular shape due to a high tyrosine content (10 tyrosines for only one tryptophan). Circular dichroism measurements spanning the far-UV region suggest that E6 contains a mixture of {alpha}-helix and random coil secondary structure elements (Figure 8BGo). A fluorescence spectrum was also recorded at an excitation wavelength of 280 nm (Figure 8CGo). The maximum of intrinsic fluorescence of E6 6C/6S was found at 331 nm, which is shifted to lower wavelength compared with the normal maximum emission of tryptophan in water of ~350 nm. This indicated that the single tryptophan of E6 6C/6S is shielded from water and buried inside the protein in a hydrophobic environment, which in turn suggests that the protein is folded. To confirm this, we performed the denaturation of E6 6C/6S by increasing the urea concentration (Figure 8DGo). Denaturation was recorded by following the exposure of the tryptophan residue to water and the resulting red shift of the maximum wavelength of re-emitted light. We observed a cooperative transition, typically found for folded proteins.



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Fig. 8. Optical characteristics of E6 6C/6S protein. (A) Comparison of ultraviolet absorption spectra of E6 6C/6S (10 µM) and MBP-E6 6C/6S (1.5 µM). (B) Circular dichroism spectrum of purified E6 6C/6S (10 µM) in the far-UV region. (C) Ultraviolet fluorescence emission spectrum of E6 6C/6S (0.5 µM) with excitation wavelength 280 nm. (D) Evolution of the maximum of fluorescence of E6 6C/6S (0.5 µM) plotted as a function of urea concentration.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The purification of high yields of active, folded and stable recombinant E6 has constituted an obstacle for the biochemical and structural characterization of this protein for 15 years. In this work, we managed to produce high yields of folded, monodisperse and stable E6 molecules. The biological activities of these samples have been checked by in vitro assays and were maintained under prolonged storage of the protein. In addition, we also obtained the first biophysical information concerning folded E6 protein.

Purification of E6 protein using GST-E6 or MBP-E6 fusions had been tried before, but led to systematic failure: after digestion of the protease-sensitive linker separating the two proteins, the GST or MBP carrier protein remained soluble, whereas the E6 moiety either precipitated or remained irreversibly bound to purification resins (Lechner and Laimins, 1994Go; Nominé et al., 2001Go). Our present work shows that this precipitation behaviour was due to the combination of two distinct processes. First, amylose resin preparations of MBP-E6 contain a majority of soluble inclusion bodies. These particles are probably formed inside the bacterial cell as soon as the fusion protein is expressed. When the crude amylose resin preparations of MBP-E6 are subjected to thrombin cleavage, the misfolded E6 polypeptide chains present in these aggregates are released and immediately precipitate. Second, a minority of E6 moieties in the amylose preparations are folded, but they are also prone to precipitation. They are extremely sensitive to redox conditions and form aggregates via intermolecular cysteine bridge formation. This aggregation probably happens only after cell breakage, during purification. Therefore, a single symptom, instability of E6 moieties after cleavage, originates from two distinct phenomena concerning two different populations of E6 molecules: aggregation of misfolded E6 moieties and aggregation of properly folded E6 moities due to oxidation.

In order to obtain stable folded E6 molecules, it was necessary to eliminate both causes of aggregation. Instead of dealing directly with the actual symptom, precipitation of E6 moieties after cleavage, we chose to optimize the quality of uncleaved MBP-E6 fusions by using monodispersity as a unique criterion. We first optimized the expression conditions. We obtained a significant decrease in the average aggregation of the MBP-E6 fusions by using expression conditions well known to decrease inclusion body formation, i.e. by lowering the expression temperature and adding a plasmid co-expressing the GroEl chaperonin. This was certainly due to a decrease in the proportion of aggregated MBP-E6 fusions correlated with the appearance of monodisperse MBP-E6 fusions. Accordingly, no monodisperse MBP-E6 protein could be isolated when we used the initial non-optimized expression conditions, whereas 20% of monodisperse MBP-E6 was isolated using the optimized expression conditions. The second step of optimization dealt with the purification conditions. We found that the two protein populations, aggregated and monodisperse, could be separated by chromatography. This could be done using either their distinct hydrophobic properties (on hydrophobic resins) or their distinct charge repartition (on ion-exchange resins). In addition, we noted the preference of the monodisperse MBP-E6 proteins for reducing conditions. This observation promoted the third phase of optimization and the engineering of a mutated E6 insensitive to redox conditions. In contrast to the MBP-E6 proteins prepared initially, the final monodisperse preparation of MBP-E6 6C/6S yielded fully soluble and folded E6 6C/6S moities when subjected to thrombin cleavage.

We were able to detect and to eliminate soluble aggregates in the preparations of MBP-E6 fusions and this allowed us to obtain soluble and active E6 protein. Can this approach be used for other fusion constructs? Carrier proteins like MBP or GST have been chosen for their high solubility. Therefore, the detection of soluble aggregates in fusion protein preparations should in general be interpreted as a symptom of aggregation of the passenger moiety. Soluble aggregates have indeed been detected in preparations of fusion proteins other than MBP-E6 and their presence always correlates with lower biological activity (Louis et al., 1991Go; Saavedra-Alanis et al., 1994Go; Lorenzo et al., 1997Go; Sachdev and Chirgwin, 1998Go, 1999Go). We propose a general strategy using monodispersity as a systematic test for checking the quality of fusion proteins and for optimizing their expression and purification. In this strategy, for each new fusion construct, a small quantity of fusion protein is first expressed and purified using a chosen initial standard condition. The aggregation rate of the soluble purified protein is then checked in a fluorimeter. If the protein is monodisperse, the purification can be pursued using standard conditions. If the protein is aggregated, several tests are rapidly performed to design the latter part of the purification. First, a buffer screening will show whether these aggregates can be disrupted upon native conditions (including reducing conditions) or whether this can be achieved only upon denaturing conditions. This will permit us to distinguish between soluble aggregates induced by folded passenger protein and soluble aggregates induced by misfolded passenger moieties. If aggregation is due to misfolded passenger moieties, priority will be given to optimization of expression conditions followed by chromatographic separation of the aggregated and monomeric fractions. If aggregation is induced by folded passenger moities, priority will be given to screening of the best purification conditions including buffers and purification steps.

Our monodispersity-based method for evaluation of quality of MBP fusions also represents a promising tool for rational modification of the sequence of recombinant proteins or protein domains. When site-directed mutagenesis is performed on a protein of unknown three-dimensional structure, it is necessary to distinguish mutations affecting the structure of the protein from mutations altering its function. Since proteins losing their capacity to fold tend to aggregate, mutations without effect on the monodispersity of the MBP-fused protein or protein domain will be interpreted as functional mutations, whereas mutations generating soluble aggregates will be interpreted as structural mutations. Our method would also be useful for studying the domain substructure of multidomain proteins. Structural or functional studies of protein domains often fail owing to uncorrect definition of the domain boundaries. When several alternative boundaries are proposed for a protein domain, the different possible fragments can be cloned as a fusion to MBP. If the MBP-fused fragment is smaller than the actual structural domain, it will undergo folding problems and expose hydrophobic patches which will promote aggregation of the fusion. If the MBP-fused fragment is larger than the actual domain, fragments of the neighbouring domains will be present and may therefore expose additional hydrophobic patches which will also promote aggregation. Minimal aggregation should be observed for the fragment which corresponds to the structural domain cloned with proper boundaries.

In this work, we used a simple and sensitive optical method to detect and quantify aggregation of fusion proteins. We have shown that this method can be applied to screen conditions favouring monodispersity of fusion proteins. This approach has allowed us to produce samples of pure, soluble and stable E6 protein. This result confirms the correlation between monodispersity and quality for fusion proteins. Our work provides guidelines for rational strategies which can be applied to optimize the recombinant production of other recalcitrant proteins or protein domains.


    Notes
 
3 To whom correspondence should be addressed.E-mail: trave{at}esbs.u-strasbg.fr Back


    Acknowledgments
 
Arnaud Pottersman is acknowledged for scientific discussions and help on dynamic light scattering, Arnt Rae and Rama Kannan for critical reading of the manuscript and scientific discussions and Élise Druhet for assistance with protein preparation. Also acknowledged are students Michael Kofler and Yann Pouliquen. Tutik Ristriani is supported by a grant from the Ligue Nationale Contre le Cancer. This work was supported by the Association pour la Recherche sur le Cancer (ARC) and by a PCV grant from CNRS, France.


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 Results
 Discussion
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Received July 7, 2000; revised November 18, 2000; accepted January 23, 2001.





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