1 Laboratoire de RMN, UPR 9003 du CNRS and 2 Laboratoire d'Immunotechnologie, UPRES 1329, École Supérieure de Biotechnologie de Strasbourg, 67400 Illkirch, France
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Abstract |
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Keywords: fusion protein/HPV E6 oncoprotein/monodispersity/proper folding/quality optimization
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Introduction |
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E6 is an oncoprotein produced by the `high-risk' human papillomaviruses (HPVs) responsible for 95% of cervical cancers (Scheffner et al., 1990). More than 100 E6-encoding DNA sequences have been published since 1985 (Chan et al., 1995
). 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., 1989
). 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, 1994
; Kukimoto et al., 1998
). 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, 1994
).
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., 2001). 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.
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Materials and methods |
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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 resinliquid 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 1824 different samples in parallel. The purified proteins were analysed by SDSPAGE 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., 1988), 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 TrisHCl 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 Trisborate 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 260400 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:
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where I280 and I340 represent the intensity of emitted light at 280 and 340 nm, respectively.
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Results |
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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., 2001). 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, 1980
). These effects can be verified by performing a 260400 nm emission scan on either MBP (monodisperse) or MBP-E6 (aggregated) diluted at the same final concentration and excited by 280 nm radiation (Figure 1A
). 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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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 1B 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 2. In the first round of optimization (Figure 2
, 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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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 2, 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., 2001). In contrast, the fraction of MBP-E6 resulting from our monodispersity optimization procedure (step 4 in Figure 2
) 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 2
, lanes 23). The HPV16 E6 sequence contains an unusually high proportion of cysteines (14 cysteines out of 158 residues) (Figure 3
). 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, 1987
). 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 3
). 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 4A
, left gel). On-resin cleavage of wild-type MBP-E6 generated E6 moieties which all remained irreversibly bound to the resin (Figure 4A
, right gel). In contrast, in-solution cleavage of the MBP-E6 6C/6S preparation yielded a fraction of soluble E6 6C/6S moieties (Figure 4B
, 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 4B
, 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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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 5A). 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 5B
). This demonstrated that the soluble E6 6C/6S moieties observed after cleavage of the amylose preparation of MBP-E6 6C/6S (Figure 4
) were actually produced by the monodisperse MBP-E6 6C/6S population.
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The observations accumulated in the previous steps enabled us to design a complete purification protocol for stable, soluble and monodisperse E6 protein (Figure 6). 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 SDSPAGE but clearly differ in their aggregation characteristics (Figure 5A
). 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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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 7A). 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., 2000
). The purified E6 6C/6S sample retained this activity, as shown by gel-retardation assay (Figure 7B
). 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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To check the purity and quality of the E6 6C/6S preparation, this sample was first subjected to UV absorption measurements (Figure 8A). 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
-helix and random coil secondary structure elements (Figure 8B
). A fluorescence spectrum was also recorded at an excitation wavelength of 280 nm (Figure 8C
). 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 8D
). 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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Discussion |
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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, 1994; Nominé et al., 2001
). 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., 1991; Saavedra-Alanis et al., 1994
; Lorenzo et al., 1997
; Sachdev and Chirgwin, 1998
, 1999
). 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.
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Notes |
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Acknowledgments |
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References |
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Received July 7, 2000; revised November 18, 2000; accepted January 23, 2001.