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EDDIE fusion proteins: Triggering autoproteolytic cleavage
Process Biochemistry 44 (2009) 1217–1224
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
EDDIE fusion proteins: Triggering autoproteolytic cleavage Rene Ueberbacher 1, Astrid Du¨rauer 1, Karin Ahrer, Sabrina Mayer, Wolfgang Sprinzl, Alois Jungbauer, Rainer Hahn * Department of Biotechnology and Austrian Center of Biopharmaceutical Technology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 April 2009 Received in revised form 18 June 2009 Accepted 23 June 2009
Heterologous proteins are often poorly expressed in Escherichia coli and especially small peptides are prone to degradation. Npro autoprotease fusion proteins, deposited as inclusion bodies in E. coli, are a versatile tool for peptide and protein overexpression and generate an authentic N terminus at the target molecule. Autoproteolytic cleavage and subsequent release of the fusion partner are initiated upon refolding. Fusion proteins with the Npro mutant EDDIE follow a monomolecular reaction. The reaction rate was only dependent on chaotrope concentration, decreasing exponentially by a factor of 1.2–1.5 for urea and by a factor of 2.1–5.3 for GuHCl. The first amino acid of the target peptide had a major impact on the reaction rate studying a set of model peptides. Reaction rates were in the range of 2.2 104 to 7.3 105 s1 and could be increased up to fivefold by exchanging the first amino acid of the target peptide. A panel of biophysical methods was used to assess EDDIE secondary and tertiary structure. Immediate formation of secondary structure and slight increase in b-sheet content of approximately 5% over the course of the cleavage reaction was observed and interpreted as aggregation. Aggregation and cleavage occurred simultaneously. EDDIE has a relatively loose structure with the cleavage site exhibiting the lowest solvent exposure. We hypothesize that this is the mechanism for establishing a spatial proximity between cleavage site and the catalytic centre of the autoprotease. Fluorescence measurements revealed that further structural changes did not occur after the initial hydrophobic collapse. Thus, the overall reaction is predominantly controlled by cleavage kinetics and refolding kinetics does not play a major role. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: EDDIE Autoprotease Cleavage kinetics Refolding Aggregation Npro
1. Introduction Recombinant proteins and peptides are often poorly expressed in Escherichia coli (E. coli), with small peptides being especially prone to intracellular degradation [1]. Therefore, several fusion tag strategies have been applied to enhance expression [2]. For proteins used for medical applications, not only is the expression yield important, but also the identity of the recombinant product with special respect to the N terminus. E. coli derived polypeptides
Abbreviations: ACN, acetonitrile; ATR-FTIR, attenuated total reflectance Fourier transform infrared; b, k0 associated parameter; c, chaotrope concentration; R2, coefficient of determination; conc., concentration; C (Cys), cysteine; GuHCl, guanidine hydrochloride; 6His, hexahistidine; HCl, hydrochloride; IB, inclusion body; k0, hypothetical maximum; k1, overall rate constant of folding and cleavage; k2, overall rate constant of misfolding; K (Lys), lysine; MTG, a-monothioglycerol; PLS-R, partial least squares regression; pep, peptide; RMSECV, root mean square error of cross-validation; RP, reversed phase; sSNEV, synthetic senescence evasion factor; t, time; TFA, trifluoroacetic acid; Tris, 2-amino-2-hydroxymethyl-propane1,3-diol; Y, yield. * Corresponding author. Tel.: +43 1 360066226; fax: +43 1 3697615. E-mail address: [email protected] (R. Hahn). 1 Both authors have contributed equally to this work. 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.06.017
are synthesised with formyl-methionine as the N-terminal amino acid [3]. Incomplete cleavage may lead to intolerable microheterogeneity of the product and even to changes in functionality and stability [4,5]. Previously, Npro fusion technology has been introduced as a novel production platform for recombinant protein expression in E. coli. This technology makes use of the autoproteolytic function of Npro derived from classical swine fever virus [6,7]. Most proteins and peptides fused to Npro are deposited as inclusion bodies (IBs), which must be dissolved under chaotropic condition. Upon switching to cosmotropic refolding conditions, the autoprotease regains activity for autoproteolytic cleavage performed at its C terminus releasing the fused partner with an authentic N terminus. By using Npro fusion technology exceptionally high titres can be achieved, even for toxic and poorly expressed proteins and peptides [8]. For preparative and industrial applications, a tailormade Npro mutant called EDDIE has been engineered (Table 1). EDDIE exhibits a better solubility as well as increased in vivo cleavage rates. In a recent study [9], we showed that refolding and autoproteolytic cleavage of EDDIE fusion proteins comprising target proteins with slow refolding kinetics, e.g. GFP, do behave
R. Ueberbacher et al. / Process Biochemistry 44 (2009) 1217–1224
1218 Table 1 Primary sequence of EDDIE.
j 1 61 121
1
11
21
31
41
51
j MELNHFELLY RDLPRKGDCR VTGSDGKLYH
j KTSKQKPVGV SGNHLGPVSG IYVEVDGEIL
j EEPVYDTAGR IYIKPGPVYY LKQAKRGTPR
j PLFGNPSEVH QDYTGPVYHR TLKWTRNTTN
j PQSTLKLPHD APLEFFDETQ CPLWVTSC
RGEDDIETTL FEETTKRIGR
60 120
Positions which had been exchanged with respect to the wild type Npro are shown in bold.
according to the commonly applied refolding model of Kiefhaber et al. [10], where refolding follows a 1st order process and aggregation, a 2nd order reaction. According to the Kiefhaber model, aggregation is the major yield-limiting factor for protein refolding. In contrast, EDDIE fusion proteins comprising short polypeptides as targets showed refolding and cleavage yield independent of protein concentration. Moreover, the reaction rate was also constant over a wide protein concentration range up to 1.0 mg ml1. Based on these observations, we proposed a reaction scheme assuming a monomolecular reaction for the refolding and autoproteolytic cleavage process whereby a misfolding reaction follows 1st order kinetics: YðtÞ ¼
k1 k1 expððk1 þ k2 Þ tÞ k1 þ k2 k1 þ k2
(1)
with Y(t) being the reaction yield as the quotient of folded (and cleaved) protein and starting protein concentration, k1 (s1) the overall rate constant of folding and cleavage, k2 (s1) the overall rate constant of misfolding, and t (s) the time. However, for protein concentrations higher than 1.0 mg ml1, the proposed model failed to properly describe the determined kinetics. This discrepancy could only partially be explained by residual urea required for dissolution of IBs in the refolding buffer. Furthermore, the fusion partner strongly influenced the refolding and cleavage kinetics of the EDDIE fusion proteins. Comparing two peptides, reaction rates differed by more than one order of magnitude. In the present study, we conducted a detailed examination of the impact of type and concentration of chaotrope and the influence of the sequence of EDDIE fusion partners on refolding and autoproteolytic cleavage of EDDIE fusion proteins. We conducted experiments with EDDIE fused to a short sixteen amino acid peptide (pep6His) consisting of a 10 amino acid spacer with a Cterminal polyhistidine tag. The sequence of the spacer was derived from the multiple cloning site of the pET30a vector used for overexpression of fusion proteins. The polyhistidine tag was introduced to ease purification and detection. Another peptide investigated was the inhibitorial peptide of senescence evasion factor (sSNEVi-C) [11]. In order to investigate the impact of the amino acid composition of the target peptide, variants of the fusion partners were designed with the first N-terminal amino acid being exchanged. The present work also focused on the correlation between batch refolding characteristics and cleavage kinetics of EDDIE, a novel protein of unknown secondary and tertiary structure. We sought a clearer understanding of the native conformation of EDDIE and of the role of EDDIE conformation changes in the autoproteolytic cleavage reaction. Initial protein folding events can occur very rapidly [12,13]. The initial hydrophobic collapse and the assembly of secondary structural elements take place in the micro- to millisecond range. Fast folding proteins even gain their native conformation in this time frame. For us, the time frame which could be accessed experimentally was between 1 and 1000 min, the time range in which refolding and cleavage kinetics were observed. The important question in this context was to elucidate whether refolding and cleavage are two independent, sequentially
occurring reactions or if they occur in parallel. Additionally, it was of interest if refolding kinetics and cleavage kinetics are interconnected. Therefore, we applied a panel of biophysical methods to follow possible structural changes during the entire refolding and cleavage process. Attenuated total reflectance-Fourier transform infrared (ATRFTIR) spectroscopy is a method frequently used for protein secondary structure analysis [14,15]. Turbidity measurements, respectively light scattering are standard techniques to study protein aggregation [16–19]. Time-resolved fluorescence studies [20], and fluorescence quenching to analyze protein structure [21] and folding as well as unfolding kinetics [22,23] are frequently applied methods in protein biochemistry. Both, tryptophan [24,25] and tyrosine [26,27] are potential targets for quenching studies. Herein, we report our observations with the aforementioned analytical methods of secondary and tertiary structure as well as aggregation analysis and interpret their outcomes in the context of the observed refolding and cleavage kinetics of various EDDIE fusion proteins. Using these data, we have attempted to establish a better understanding of the EDDIE cleavage mechanism on a molecular level. 2. Materials and methods 2.1. Protein and buffer preparation The recombinant fusion proteins EDDIE-pep6His, EDDIE-K-pep6His, EDDIEsSNEVi-C and EDDIE-C-sSNEVi were overexpressed in E. coli strain BL21 (DE3) with a pET30a plasmid (Novagen, Madison, WI, USA) containing the respective coding gene [8]. E. coli fed batch cultivations were conducted with semi-synthetic medium on a 5 l scale according to Clementschitsch et al. [28]. IBs were harvested by centrifugation, followed by a number of washing steps with water and detergents. Details have been described by Kaar et al. [9]. IBs were dissolved in a buffer containing 10.0 M urea, 50 mM Tris, and 100 mM MTG at pH 7.3. Urea and Tris were purchased from Merck (Darmstadt, Germany), MTG from Sigma–Aldrich (Vienna, Austria). The dissolved IBs were then centrifuged for 30 min at 16,110 g and 277 K. Insoluble components were removed by subsequent filtration through 0.4 and 0.22 mm filters (Millipore, Billerica, USA). Protein concentration was determined with a Cary 50 Bio UV–vis spectrophotometer (Varian, Palo Alto, USA) at 280 nm. 2.2. Protein refolding Protein refolding was performed as previously described [9]. Refolding of the IB protein was initiated by rapid dilution into refolding buffer containing 1 M Tris, 0.25 M D(+)-sucrose, 2 mM EDTA, 20 mM MTG at pH 7.3 and residual levels of chaotropic agents from dissolution buffer. D(+)-Sucrose was purchased from Acros Organics (New Jersey, USA). Since the protein concentration for refolding experiments was determined by adding the solution of dissolved IB into the refolding buffer, the amount of residual chaotropic reagent increased with increasing protein concentration. Exceptions were experiments investigating the influence of type and concentration of chaotrope, where refolding buffers with defined chaotrope concentrations independent of the protein concentration were used. Refolding samples were incubated at 291 K without further stirring. Immediately after transfer of protein to refolding buffer, an aliquot of the refolding sample was subjected to RP-HPLC analysis, performed at distinct time intervals from the same vial to follow the time course of refolding. 2.3. RP-HPLC analysis Protein analyses by RP-HPLC were performed with a JupiterTM C-4 column (2 mm 150 mm, 5 mm, 300 A˚) (Phenomenex, Torrance, CA, USA) on Agilent 1100 series chromatographic system (Waldbronn, Germany) using an additional SecurityGuardTM-cartridge. A buffer system of 0.1% (v/v) TFA, 5% (v/v) ACN
R. Ueberbacher et al. / Process Biochemistry 44 (2009) 1217–1224 (LiChrosolv, Merck, Darmstadt, Germany) in water as buffer A and 0.1% (v/v) TFA in ACN as buffer B was used. Refolding samples were directly injected; elution was performed with gradients differing in steepness depending on the nature of the cleavage products to be analysed. Detection proceeded at two wavelengths, 214 and 280 nm to allow distinguishing between proteins and buffer components. Calibration curves for EDDIE-pep6His, EDDIE-sSNEVi-C, pep6His, and sSNEVi-C were established to allow quantification of IB protein and cleavage products. 2.4. ATR-FTIR ATR-FTIR measurements were performed with a Vertex 70 FTIR spectrometer with a liquid N2-cooled MCT detector and a BioATRCell II from Bruker Optics (Vienna, Austria). The BioATRCell II was purged with dry air and tempered at 25 rpm 0.1 K. Spectra were collected from 4000 to 850 cm1 with a resolution of 4 cm1 and 64 scans were averaged for each spectrum. For each experiment, a 25 ml aliquot of sample was used. For protein samples, a concentration of 2.0 mg ml1 was used. A multivariate analysis technique was used to determine protein secondary structure of EDDIE [25]. Reference spectra of 26 model proteins of known crystal structures were recorded. We selected the model proteins to have representative data from numerous proteins of different secondary structure compositions. A partial least squares regression (PLS-R) method was applied to develop an empirical model for secondary structure prediction based on the set of reference spectra. The PLS-R algorithms for calibration and prediction implemented in the spectroscopic software OPUS QUANT2 by Bruker were used. A model using cross-validation was applied for validation. The validation of the model used for prediction of a-helix yielded a root mean square error of cross-validation (RMSECV) of 4.6% and an R2 value of 95.9%. For the prediction of b-sheet, a RMSECV of 4.71% and a R2 of 89.4% was determined. We based the model used for prediction of secondary structure on the assumption that proteins consist of a-helix, b-sheet, and turn/unordered structures. 2.5. Turbidity Turbidity was measured with a Cary 50 Bio UV–vis spectrophotometer (Varian, Palo Alto, USA) at 350 nm. Measurements were performed over the whole timescale used to study cleavage kinetics at a protein concentration of 2.0 mg ml1. 2.6. Fluorescence Fluorescence measurements were performed with a Cary Eclipse spectrofluorimeter (Varian, Palo Alto, USA) equipped with a thermostatically controlled cuvette holder. Excitation was at either 271 or 295 nm. Emission, for both excitation wavelengths, was detected at 303, 338, and 352 nm. Fluorescence was measured at a protein concentration of 2.0 mg ml1.
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protein concentrations of 0.4 mg ml1. For protein concentration of 2.0 mg ml1, the minimal adjustable chaotrope concentration was between 0.2 and 0.5 M depending on chaotrope and fusion protein. The recorded kinetics were based on the detection of released peptide by RP-HPLC. However, proper refolding of EDDIE is a prerequisite for the autoproteolytic cleavage. Thus, at this point we prefer the term refolding and cleavage kinetics to emphasize that two different reactions are associated. It is important to point out that the respective rate constants represent apparent refolding and cleavage constants because the time course of the reactions and their interdependence is unknown and are one objective of this study, respectively. Representative refolding and cleavage time courses for EDDIE-pep6His at 0.4 and 2.0 mg ml1 are shown in Fig. 1. We observed a range of tolerable chaotrope concentrations where the ultimate cleavage yield was almost constant. The tolerable concentrations varied for the two chaotropes. For urea, the yield remained constant up to 1.4 M, whereas a significant yield reduction occurred at GuHCl concentrations higher than 0.7 M. This outcome is consistent with the report that GuHCl acts as a stronger denaturant than urea [29]. Concentrations higher than 1.2 M GuHCl led to full inhibition of autoproteolytic cleavage, whereas at urea concentration as high as 2.0 M, a yield of 30% released peptide could still be determined for the time frame investigated. The calculated rate constant k1, determined by fitting Eq. (1) to these data, decreased with increasing concentration of both chaotropic reagents. The entire data set in terms of k1 representing the influence of chaotrope concentration on cleavage for EDDIE-pep6His and EDDIE-sSNEVi-C is shown in Fig. 2. The graphs shown represent data comprising fixed protein concentrations of 0.4 and 2.0 mg ml1 with varying chaotrope concentrations (shown in Fig. 1) as well as a concentration series of 0.1–2.0 mg ml1 with chaotrope concentrations resulting from the dilution step. The decline of k1 was approximated by an exponential function in the form of: k1 ¼ k0 expðb cÞ
(2)
3. Results and discussion 3.1. Influence of chaotrope and protein concentration on refolding and cleavage of EDDIE fusion proteins The most relevant molecular characteristics of EDDIE, the fusion partners, and the respective fusion proteins are summarised in Table 2. For investigating the influence of type and concentration of chaotrope on refolding and cleavage of EDDIE fusion proteins, cleavage kinetics were determined at two concentrations for EDDIE-pep6His and EDDIE-sSNEVi-C: 0.4 and 2.0 mg ml1. These experiments were performed in the presence of increasing concentrations of urea and GuHCl in the refolding buffer. A range of 0.12–2.0 M urea and 0.06–2.0 M GuHCl final chaotrope concentration during refolding and cleavage was investigated for the fusion proteins EDDIE-pep6His and EDDIE-sSNEVi-C at
where k0 represents a hypothetical maximal rate constant, b a parameter for the decrease of the rate constant and c the chaotrope concentration. A summary of the estimated parameters is shown in Table 3. This data analysis showed that refolding and cleavage kinetics are in fact independent of protein concentration as proposed by Eq. (1) and only depend on the chaotrope concentration. As expected, this effect is more pronounced for GuHCl and represented by low values of the parameter b. Interestingly, k1 differs by one order of magnitude for the two target peptides regardless of the chaotrope system, although both represent a low molecular weight polypeptide without a distinct structure. This fact suggests dominant influence of the type of target peptide and possibly the first amino acid of the target peptide on the refolding and cleavage rate.
Table 2 Biophysical properties of peptides used for EDDIE fusion proteins. EDDIE and fusion partnersa
Amino acid sequence
pI of peptide/EDDIE fusion protein
Number of cysteines
Number of amino acids
Molecular mass (kDa)b
EDDIE pep6His K-pep6His sSNEVi-C C-sSNEVi
see Table 1 SVDKLAAALEHHHHHH KSVDKLAAALEHHHHHH KVAHPIRPKPPSATSIPAIC CKVAHPIRPKPPSATSIPAI
6.59 6.55/6.6 7.2/6.86 9.5/8.29 9.5/8.29
3 0 0 1 1
168 16 17 20 20
19.1 1.8 1.9 2.0 2.0
a
Proteins derived from codon-optimised genes are preceded by an ‘s’.
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Fig. 1. Influence of chaotrope on autoproteolysis of EDDIE-pep6His in the presence of increasing concentrations of urea (A and C) and GuHCl (B and D). Data points represent experimental values determined by HPLC analysis. Lines represent fits with Eq. (1). The protein concentration was 0.4 mg ml1 in Fig. 1A and B and 2.0 mg ml1 in Fig. 1C and D. Autoproteolysis of EDDIE fusion proteins was started by dilution of IB solutions in refolding buffer adjusting the according protein concentration. The residual chaotrope concentration represents the minimal concentration for refolding which is adjustable by dilution. Higher chaotrope concentrations were achieved by addition of chaotrope to the refolding buffer.
3.2. Influence of 1st amino acid of target peptide on refolding and cleavage of EDDIE fusion proteins
Fig. 2. Influence of chaotrope concentration on cleavage kinetics of EDDIE-pep6His and EDDIE-sSNEVi-C. Symbols represent rate constants k1 at different chaotrope and protein concentrations (0.1–2.0 mg ml1) determined by Eq. (1). Lines represent curve fits with an exponential function according to Eq. (2). Global fits were carried out for each data set comprising fixed protein concentrations of 0.4 and 2.0 mg ml1 with varying chaotrope concentrations as well as a concentration series of 0.1–2.0 mg ml1 with chaotrope concentrations resulting from the dilution step.
The refolding and cleavage kinetics of four selected EDDIE fusion proteins were investigated at four different protein concentrations ranging from 0.1 to 2.0 mg ml1 (Fig. 3): EDDIEpep6His, EDDIE-K-pep6His, EDDIE-sSNEVi-C and EDDIE-C-sSNEVi. EDDIE-pep6His and EDDIE-K-pep6His as well as EDDIE-sSNEVi-C and EDDIE-C-sSNEVi only differ in the first amino acid of the target peptides (Table 2). At protein concentrations up to 2.0 mg ml1, the yield of released peptide is independent of protein concentration and could be described properly by the proposed model for autoproteolysis of EDDIE fusion proteins defined by Eq. (1). The plots of normalised cleavage yield vs. time showed that reaction rate is strongly influenced by the type of fusion partner and the nature of its first amino acid. Insertion of Lys as the first amino acid in pep6His led to a fivefold decrease of the rate constant (3.7 104 to 7.3 105 s1) (Fig. 3A and B). Transferring Cys from the C terminus to the N terminus led to a ninefold increase of the rate constant for autoproteolytic cleavage of the EDDIE fusion proteins with the target peptide sSNEVi (2.5 105 to 2.2 104 s1) (Fig. 3C and D). Thus, for these fusion proteins, the first amino acid at the N terminus of the fusion partner has a predominant
R. Ueberbacher et al. / Process Biochemistry 44 (2009) 1217–1224
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Table 3 Parameters calculated with Eq. (2) for chaotrope influence on autoproteolysis rate of EDDIE-pep6His and EDDIE-sSNEVi-C. k0 (s1) is a hypothetical maximal rate constant and b (M1) a parameter for the decrease of the rate constant. EDDIE-pep6His Urea 1
k0 (s ) b (M1)
(4.5 0.2) 10 1.5 0.1
EDDIE-SNEVi-C GuHCl
4
(2.2 0.4) 10 5.3 0.8
Urea 3
(4.9 0.5) 10 1.2 0.2
GuHCl 5
(8.3 1.8) 105 2.1 0.6
Fig. 3. Autoproteolysis of EDDIE fusion proteins. Kinetics of cleavage with increasing concentrations of EDDIE-pep6His (A), EDDIE-K-pep6His (B), EDDIE-sSNEVi-C (C) and EDDIE-C-sSNEVi (D) were determined by RP-HPLC analysis as described in Section 2.
influence on the reaction rate. As also demonstrated in Fig. 3, the exponential dependence of k1 on the chaotrope concentration resulted in substantially longer times for the systems with slow kinetics to reach equilibrium. Thus, it takes longer time to reach maximum yield when autoproteolysis is performed with high protein concentrations which carry higher levels of residual chaotropic reagents. In order to investigate how the refolding and cleavage are timely interconnected, we followed structural changes of the fusion proteins over time by biophysical methods. 3.3. Analysis of EDDIE secondary structure by ATR-FTIR spectroscopy By the first measurement, performed 1 min after the start of refolding, secondary structure was already evident in all cases. However, the secondary structure composition of the 4 EDDIE
fusion proteins changed with time. This is exemplarily shown for EDDIE-pep6His in Fig. 4. The maximum of the amide I band shifted to lower wavenumbers and the relative area of the peaks corresponding to b-sheets (1635–1615 cm1) increased. On the other hand the amount of a-helix decreased with time. It has been shown for several proteins that an increase of b-sheet structures, often accompanied by a loss of a-helix, correlates with aggregation [20,23]. Militello et al. attributed the peak at 1620 cm1 to intramolecular b-sheets involved in aggregation [22]. Zandomeneghi et al. also showed that a shift of the maximum to lower wavenumbers and a peak at 1620 cm1 are characteristic for intramolecular b-sheets [30]. For all EDDIE fusion proteins we observed a decrease of a-helix and concomitant increase of b-sheet structures (Table 4). The whole data set is shown in supplementary Fig. 1. Importantly, no direct link between changes in secondary structure over time and refolding and cleavage
R. Ueberbacher et al. / Process Biochemistry 44 (2009) 1217–1224
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Fig. 4. Resolution of secondary structures in EDDIE-pep6His amide I bands at start and end point of refolding and cleavage reaction. Fourier self-deconvolution (FSD) and Gaussian curve-fitting of the amide I band was performed to resolve the respective subbands. The subband at 1618 cm1 corresponds to intermolecular b-sheets while the subband at approximately 1635 cm1 corresponds to intramolecular b-sheets.
kinetics could be observed. Therefore, the different kinetics of the four EDDIE fusion proteins cannot be attributed to structural rearrangements based on analysis of secondary structure. 3.4. Analysis of EDDIE aggregation by turbidity measurements Clearly, EDDIE formed aggregates in all cases (Fig. 5A). Absorbance started to increase immediately after establishing refolding conditions. The turbidity data was related to refolding and cleavage kinetics data (Fig. 5B). For EDDIE fusion proteins which showed fast reaction kinetics, aggregation occurred faster, Table 4 Determination of a-helix and b-sheet content of EDDIE-pep6His, EDDIE-K-pep6His, EDDIE-sSNEVi-C and EDDIE-C-sSNEVi over the time course of the refolding and cleavage process by ATR-FTIR.
Fig. 5. (A) Turbidity measurements of EDDIE fusion proteins under batch refolding conditions and (B) cleavage kinetics determined by RP-HPLC and fit to 1st order refolding model (Eq. (1)). The protein concentration for studies with EDDIE-sSNEVi-C, EDDIE-C-sSNEVi EDDIE-pep6His and EDDIE-K-pep6His was 2 mg ml1.
leading to an increase in scattered light at 350 nm. In earlier work, we stated that aggregation was not a rate limiting factor for the autoproteolytic cleavage of EDDIE fusion proteins [9]. According to turbidity data, aggregation showed the same progression as refolding and cleavage kinetics. Aggregation and release of peptide appeared to be almost parallel events, indicating that aggregation took place right after cleavage. We reasoned that EDDIE had a lower tendency to aggregate when fused to sSNEVi and pep6His variants. The relatively strong aggregation process also suggests that the increase of b-sheet content of EDDIE discussed in the ATR-FTIR section was mainly caused by the aggregation preceding formation of b-sheet structures.
Time (min)
a-Helix (%)
b-Sheet (%)
EDDIE-pep6His
1 30 1000
20.7 18.9 15.0
23.2 24.8 27.3
3.5. Analysis of EDDIE aggregation and tertiary structure by fluorescence spectroscopy
EDDIE-K-pep6His
1 60 1000
17.6 15.6 14.6
23.4 26.0 28.5
EDDIE-sSNEVi-C
1 40 1200
25.2 13.8 12.6
22.7 23.4 29.5
EDDIE-C-sSNEVi
1 60 1200
15.2 9.6 8.5
27.0 29.1 31.0
EDDIE has a known sequence of 168 amino acids [8] and is a well suited protein for fluorescence studies. It contains 9 Tyr residues, Tyr10, Tyr25, Tyr82, Tyr89, Tyr90, Tyr93, Tyr98, Tyr129, Tyr132, scattered throughout the entire sequence except the C terminus. In close proximity to the C terminus, where cleavage takes place, 2 Trp residues are located, Trp154 and Trp164 (Table 1). The sites of these residues offered the possibility to selectively gain information about changes of the whole protein structure vs. the C-terminal region. Tyr and Trp residues were both excited at 271 nm. For selective excitation of Trp residues only, we used an
Protein concentration was 2 mg ml1.
R. Ueberbacher et al. / Process Biochemistry 44 (2009) 1217–1224
excitation wavelength of 295 nm. The fusion partners pep6His and sSNEVi do not contain Phe, Tyr or Trp residues, and thus will not contribute to fluorescence emission. The difference of the Trp fluorescence spectrum of EDDIE between dissolved IBs (10 M urea, unfolded) and under refolding conditions was initially investigated using the EDDIE-pep6His fusion protein. Trp fluorescence is highly dependent on solvent exposure. Generally speaking, solvent exposed Trp emits at 340– 355 nm, whereas those residues buried in the interior of the folded protein fluoresce at shorter wavelengths, typically between 308 and 340 nm [31]. The emission maximum for unfolded EDDIE was determined to be at 352 nm. Under refolding conditions, the maximum shifted to 338 nm (data not shown). An emission maximum at 338 nm is close to the upper limit of 340 nm for native Trp fluorescence described in the literature [32], but is also within the limits of fluorescence for a Trp residue that is in a solvent-shielded, hydrophobic environment. Less solvent exposure results in an emission maximum shifted to shorter wavelengths. Consequently, a relatively high exposure of Trp residues to the solvent in the native state results in an emission maximum at longer wavelengths. No further significant shifts of the Trp emission maxima were observed over the extended times associated with cleavage for sSNEVi and pep6His variants. In conjunction with the results obtained from ATR-FTIR and turbidity measurements, our observations indicate that no conformational changes took place after the collapse of the protein once refolding conditions were established. To examine whether additional folding events occurred during the extended time frame of batch refolding, we investigated the ratio of emission for folded and unfolded EDDIE. Thus, we excited at 295 and 271 nm and measured emission at wavelengths 338 and 352 nm. Over the whole time range, the ratio of fluorescence emissions was constant (supplementary Fig. 2). These results indicate that neither Tyr nor Trp residues were associated with structural changes of EDDIE. Therefore, we assume that no significant structural changes occur over the observed time frame. Fluorescence intensity showed a time dependent decrease. Typical quenching data is exemplarily shown for EDDIE-sSNEVi-C in Fig. 6. Data for the other EDDIE fusion proteins is depicted in supplementary Fig. 3. This raised the question whether aggregation processes came into play. Quenching is a contact phenomenon and since no chemicals with the potential to quench were added
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during the course of refolding, the decrease of fluorescence intensity must have been caused by aggregation. The shape of the curves in Fig. 6 allowed us to draw conclusions regarding the fluorophore exposure to the solvent [32]. Quenching of fluorescence attributed to exciting Trp residues was less pronounced and intensity decreased linearly. Greater quenching of Tyr fluorescence occurred and intensity decreased rather hyperbolically. This can be explained by the fact that Trp residues were less surface accessible than Tyr residues. Trp residues were already shown to have a relatively moderate solvent exposure since native EDDIE emits at 338 nm (close to upper limit of 340 nm for native Trp fluorescence). This indicated that EDDIE was in a rather loose and flexible state. The quenching studies indicate that the C-terminal Trp residues in close vicinity to the cleavage site are more shielded from the solvent than the Tyr residues. Combining results obtained by fluorescence measurements, we propose that at the level of tertiary structure, native EDDIE is a rather unstructured protein. Importantly, the cleavage site has the highest solvent shielding which may be explained by the formation of a cavity. So far, NMR and X-ray crystallographic experiments have failed in providing a 3D-structure, which may be due to this loose conformation. Ru¨menapf et al. showed that Cys69 is essential for cleavage [6]. Our results support the findings of Ru¨menapf et al. We hypothesise that the mechanism for establishing a spatial proximity between the cleavage site and the catalytic centre of the autoprotease is the formation of a cavity comprising the folding of the C terminus towards Cys69. In summary, all applied biophysical methods did not detect significant structural changes of fusion proteins during the examined time course of autoproteolytic cleavage. Since the proper refolding of EDDIE is the prerequisite for cleavage, these results strongly indicate that refolding of the autoprotease is already completed at the first measurement. According to that, the kinetics recorded for the autoproteolytic process were largely dominated by the cleavage reaction. This is further supported by the fact, that no significant differences in secondary structure could be determined for the different fusion proteins although the reaction rates of their corresponding autoproteolytic cleavage differed up to one order of magnitude. Herein, the impact of the 1st amino acid dominated over the amino acid composition of the respective target peptide. This suggests that the cleavage rate and thus the overall kinetics of the autoproteolytic reaction are determined by the steric configuration of the cleavage site. Our investigations corroborate the model we previously developed which suggested a monomolecular reaction for autoproteolysis of EDDIE fusion proteins [9]. In fact, yield and reaction rate are independent of protein concentration. This implicates, that the initial refolding reaction preceding cleavage is also independent of the protein concentration but depends on type and concentration of chaotrope. This represents a remarkable property of EDDIE. The findings presented here allow prediction and control of preparative production of peptides using the EDDIE autoprotease fusion system. However, resolution of its atomic structure will be necessary to fully understand the cleavage mechanism. Acknowledgements
Fig. 6. Fluorescence quenching data for EDDIE-sSNEVi-C under refolding conditions. Protein fluorescence was excited at 271 and 295 nm, emission was measured at 338 nm and a protein concentration of 2 mg ml1 was used.
This work was carried out in the Austrian Centre of Biopharmaceutical Technology which is a Competence Centre within the Kind Knet programme funded by the Austrian Federal Ministry of Economics and Labour (FWF) and the provinces Vienna and Tyrol. We thank Prof. Christian Obinger and Dr. Manfred Schwanninger, Department of Chemistry at the University of Natural Resources and Applied Life Sciences Vienna, for their valuable support with respect to spectroscopic methods.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.procbio.2009.06.017.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
EDDIE fusion proteins: Triggering autoproteolytic cleavage Rene Ueberbacher 1, Astrid Du¨rauer 1, Karin Ahrer, Sabrina Mayer, Wolfgang Sprinzl, Alois Jungbauer, Rainer Hahn * Department of Biotechnology and Austrian Center of Biopharmaceutical Technology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 April 2009 Received in revised form 18 June 2009 Accepted 23 June 2009
Heterologous proteins are often poorly expressed in Escherichia coli and especially small peptides are prone to degradation. Npro autoprotease fusion proteins, deposited as inclusion bodies in E. coli, are a versatile tool for peptide and protein overexpression and generate an authentic N terminus at the target molecule. Autoproteolytic cleavage and subsequent release of the fusion partner are initiated upon refolding. Fusion proteins with the Npro mutant EDDIE follow a monomolecular reaction. The reaction rate was only dependent on chaotrope concentration, decreasing exponentially by a factor of 1.2–1.5 for urea and by a factor of 2.1–5.3 for GuHCl. The first amino acid of the target peptide had a major impact on the reaction rate studying a set of model peptides. Reaction rates were in the range of 2.2 104 to 7.3 105 s1 and could be increased up to fivefold by exchanging the first amino acid of the target peptide. A panel of biophysical methods was used to assess EDDIE secondary and tertiary structure. Immediate formation of secondary structure and slight increase in b-sheet content of approximately 5% over the course of the cleavage reaction was observed and interpreted as aggregation. Aggregation and cleavage occurred simultaneously. EDDIE has a relatively loose structure with the cleavage site exhibiting the lowest solvent exposure. We hypothesize that this is the mechanism for establishing a spatial proximity between cleavage site and the catalytic centre of the autoprotease. Fluorescence measurements revealed that further structural changes did not occur after the initial hydrophobic collapse. Thus, the overall reaction is predominantly controlled by cleavage kinetics and refolding kinetics does not play a major role. ß 2009 Elsevier Ltd. All rights reserved.
Keywords: EDDIE Autoprotease Cleavage kinetics Refolding Aggregation Npro
1. Introduction Recombinant proteins and peptides are often poorly expressed in Escherichia coli (E. coli), with small peptides being especially prone to intracellular degradation [1]. Therefore, several fusion tag strategies have been applied to enhance expression [2]. For proteins used for medical applications, not only is the expression yield important, but also the identity of the recombinant product with special respect to the N terminus. E. coli derived polypeptides
Abbreviations: ACN, acetonitrile; ATR-FTIR, attenuated total reflectance Fourier transform infrared; b, k0 associated parameter; c, chaotrope concentration; R2, coefficient of determination; conc., concentration; C (Cys), cysteine; GuHCl, guanidine hydrochloride; 6His, hexahistidine; HCl, hydrochloride; IB, inclusion body; k0, hypothetical maximum; k1, overall rate constant of folding and cleavage; k2, overall rate constant of misfolding; K (Lys), lysine; MTG, a-monothioglycerol; PLS-R, partial least squares regression; pep, peptide; RMSECV, root mean square error of cross-validation; RP, reversed phase; sSNEV, synthetic senescence evasion factor; t, time; TFA, trifluoroacetic acid; Tris, 2-amino-2-hydroxymethyl-propane1,3-diol; Y, yield. * Corresponding author. Tel.: +43 1 360066226; fax: +43 1 3697615. E-mail address: [email protected] (R. Hahn). 1 Both authors have contributed equally to this work. 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.06.017
are synthesised with formyl-methionine as the N-terminal amino acid [3]. Incomplete cleavage may lead to intolerable microheterogeneity of the product and even to changes in functionality and stability [4,5]. Previously, Npro fusion technology has been introduced as a novel production platform for recombinant protein expression in E. coli. This technology makes use of the autoproteolytic function of Npro derived from classical swine fever virus [6,7]. Most proteins and peptides fused to Npro are deposited as inclusion bodies (IBs), which must be dissolved under chaotropic condition. Upon switching to cosmotropic refolding conditions, the autoprotease regains activity for autoproteolytic cleavage performed at its C terminus releasing the fused partner with an authentic N terminus. By using Npro fusion technology exceptionally high titres can be achieved, even for toxic and poorly expressed proteins and peptides [8]. For preparative and industrial applications, a tailormade Npro mutant called EDDIE has been engineered (Table 1). EDDIE exhibits a better solubility as well as increased in vivo cleavage rates. In a recent study [9], we showed that refolding and autoproteolytic cleavage of EDDIE fusion proteins comprising target proteins with slow refolding kinetics, e.g. GFP, do behave
R. Ueberbacher et al. / Process Biochemistry 44 (2009) 1217–1224
1218 Table 1 Primary sequence of EDDIE.
j 1 61 121
1
11
21
31
41
51
j MELNHFELLY RDLPRKGDCR VTGSDGKLYH
j KTSKQKPVGV SGNHLGPVSG IYVEVDGEIL
j EEPVYDTAGR IYIKPGPVYY LKQAKRGTPR
j PLFGNPSEVH QDYTGPVYHR TLKWTRNTTN
j PQSTLKLPHD APLEFFDETQ CPLWVTSC
RGEDDIETTL FEETTKRIGR
60 120
Positions which had been exchanged with respect to the wild type Npro are shown in bold.
according to the commonly applied refolding model of Kiefhaber et al. [10], where refolding follows a 1st order process and aggregation, a 2nd order reaction. According to the Kiefhaber model, aggregation is the major yield-limiting factor for protein refolding. In contrast, EDDIE fusion proteins comprising short polypeptides as targets showed refolding and cleavage yield independent of protein concentration. Moreover, the reaction rate was also constant over a wide protein concentration range up to 1.0 mg ml1. Based on these observations, we proposed a reaction scheme assuming a monomolecular reaction for the refolding and autoproteolytic cleavage process whereby a misfolding reaction follows 1st order kinetics: YðtÞ ¼
k1 k1 expððk1 þ k2 Þ tÞ k1 þ k2 k1 þ k2
(1)
with Y(t) being the reaction yield as the quotient of folded (and cleaved) protein and starting protein concentration, k1 (s1) the overall rate constant of folding and cleavage, k2 (s1) the overall rate constant of misfolding, and t (s) the time. However, for protein concentrations higher than 1.0 mg ml1, the proposed model failed to properly describe the determined kinetics. This discrepancy could only partially be explained by residual urea required for dissolution of IBs in the refolding buffer. Furthermore, the fusion partner strongly influenced the refolding and cleavage kinetics of the EDDIE fusion proteins. Comparing two peptides, reaction rates differed by more than one order of magnitude. In the present study, we conducted a detailed examination of the impact of type and concentration of chaotrope and the influence of the sequence of EDDIE fusion partners on refolding and autoproteolytic cleavage of EDDIE fusion proteins. We conducted experiments with EDDIE fused to a short sixteen amino acid peptide (pep6His) consisting of a 10 amino acid spacer with a Cterminal polyhistidine tag. The sequence of the spacer was derived from the multiple cloning site of the pET30a vector used for overexpression of fusion proteins. The polyhistidine tag was introduced to ease purification and detection. Another peptide investigated was the inhibitorial peptide of senescence evasion factor (sSNEVi-C) [11]. In order to investigate the impact of the amino acid composition of the target peptide, variants of the fusion partners were designed with the first N-terminal amino acid being exchanged. The present work also focused on the correlation between batch refolding characteristics and cleavage kinetics of EDDIE, a novel protein of unknown secondary and tertiary structure. We sought a clearer understanding of the native conformation of EDDIE and of the role of EDDIE conformation changes in the autoproteolytic cleavage reaction. Initial protein folding events can occur very rapidly [12,13]. The initial hydrophobic collapse and the assembly of secondary structural elements take place in the micro- to millisecond range. Fast folding proteins even gain their native conformation in this time frame. For us, the time frame which could be accessed experimentally was between 1 and 1000 min, the time range in which refolding and cleavage kinetics were observed. The important question in this context was to elucidate whether refolding and cleavage are two independent, sequentially
occurring reactions or if they occur in parallel. Additionally, it was of interest if refolding kinetics and cleavage kinetics are interconnected. Therefore, we applied a panel of biophysical methods to follow possible structural changes during the entire refolding and cleavage process. Attenuated total reflectance-Fourier transform infrared (ATRFTIR) spectroscopy is a method frequently used for protein secondary structure analysis [14,15]. Turbidity measurements, respectively light scattering are standard techniques to study protein aggregation [16–19]. Time-resolved fluorescence studies [20], and fluorescence quenching to analyze protein structure [21] and folding as well as unfolding kinetics [22,23] are frequently applied methods in protein biochemistry. Both, tryptophan [24,25] and tyrosine [26,27] are potential targets for quenching studies. Herein, we report our observations with the aforementioned analytical methods of secondary and tertiary structure as well as aggregation analysis and interpret their outcomes in the context of the observed refolding and cleavage kinetics of various EDDIE fusion proteins. Using these data, we have attempted to establish a better understanding of the EDDIE cleavage mechanism on a molecular level. 2. Materials and methods 2.1. Protein and buffer preparation The recombinant fusion proteins EDDIE-pep6His, EDDIE-K-pep6His, EDDIEsSNEVi-C and EDDIE-C-sSNEVi were overexpressed in E. coli strain BL21 (DE3) with a pET30a plasmid (Novagen, Madison, WI, USA) containing the respective coding gene [8]. E. coli fed batch cultivations were conducted with semi-synthetic medium on a 5 l scale according to Clementschitsch et al. [28]. IBs were harvested by centrifugation, followed by a number of washing steps with water and detergents. Details have been described by Kaar et al. [9]. IBs were dissolved in a buffer containing 10.0 M urea, 50 mM Tris, and 100 mM MTG at pH 7.3. Urea and Tris were purchased from Merck (Darmstadt, Germany), MTG from Sigma–Aldrich (Vienna, Austria). The dissolved IBs were then centrifuged for 30 min at 16,110 g and 277 K. Insoluble components were removed by subsequent filtration through 0.4 and 0.22 mm filters (Millipore, Billerica, USA). Protein concentration was determined with a Cary 50 Bio UV–vis spectrophotometer (Varian, Palo Alto, USA) at 280 nm. 2.2. Protein refolding Protein refolding was performed as previously described [9]. Refolding of the IB protein was initiated by rapid dilution into refolding buffer containing 1 M Tris, 0.25 M D(+)-sucrose, 2 mM EDTA, 20 mM MTG at pH 7.3 and residual levels of chaotropic agents from dissolution buffer. D(+)-Sucrose was purchased from Acros Organics (New Jersey, USA). Since the protein concentration for refolding experiments was determined by adding the solution of dissolved IB into the refolding buffer, the amount of residual chaotropic reagent increased with increasing protein concentration. Exceptions were experiments investigating the influence of type and concentration of chaotrope, where refolding buffers with defined chaotrope concentrations independent of the protein concentration were used. Refolding samples were incubated at 291 K without further stirring. Immediately after transfer of protein to refolding buffer, an aliquot of the refolding sample was subjected to RP-HPLC analysis, performed at distinct time intervals from the same vial to follow the time course of refolding. 2.3. RP-HPLC analysis Protein analyses by RP-HPLC were performed with a JupiterTM C-4 column (2 mm 150 mm, 5 mm, 300 A˚) (Phenomenex, Torrance, CA, USA) on Agilent 1100 series chromatographic system (Waldbronn, Germany) using an additional SecurityGuardTM-cartridge. A buffer system of 0.1% (v/v) TFA, 5% (v/v) ACN
R. Ueberbacher et al. / Process Biochemistry 44 (2009) 1217–1224 (LiChrosolv, Merck, Darmstadt, Germany) in water as buffer A and 0.1% (v/v) TFA in ACN as buffer B was used. Refolding samples were directly injected; elution was performed with gradients differing in steepness depending on the nature of the cleavage products to be analysed. Detection proceeded at two wavelengths, 214 and 280 nm to allow distinguishing between proteins and buffer components. Calibration curves for EDDIE-pep6His, EDDIE-sSNEVi-C, pep6His, and sSNEVi-C were established to allow quantification of IB protein and cleavage products. 2.4. ATR-FTIR ATR-FTIR measurements were performed with a Vertex 70 FTIR spectrometer with a liquid N2-cooled MCT detector and a BioATRCell II from Bruker Optics (Vienna, Austria). The BioATRCell II was purged with dry air and tempered at 25 rpm 0.1 K. Spectra were collected from 4000 to 850 cm1 with a resolution of 4 cm1 and 64 scans were averaged for each spectrum. For each experiment, a 25 ml aliquot of sample was used. For protein samples, a concentration of 2.0 mg ml1 was used. A multivariate analysis technique was used to determine protein secondary structure of EDDIE [25]. Reference spectra of 26 model proteins of known crystal structures were recorded. We selected the model proteins to have representative data from numerous proteins of different secondary structure compositions. A partial least squares regression (PLS-R) method was applied to develop an empirical model for secondary structure prediction based on the set of reference spectra. The PLS-R algorithms for calibration and prediction implemented in the spectroscopic software OPUS QUANT2 by Bruker were used. A model using cross-validation was applied for validation. The validation of the model used for prediction of a-helix yielded a root mean square error of cross-validation (RMSECV) of 4.6% and an R2 value of 95.9%. For the prediction of b-sheet, a RMSECV of 4.71% and a R2 of 89.4% was determined. We based the model used for prediction of secondary structure on the assumption that proteins consist of a-helix, b-sheet, and turn/unordered structures. 2.5. Turbidity Turbidity was measured with a Cary 50 Bio UV–vis spectrophotometer (Varian, Palo Alto, USA) at 350 nm. Measurements were performed over the whole timescale used to study cleavage kinetics at a protein concentration of 2.0 mg ml1. 2.6. Fluorescence Fluorescence measurements were performed with a Cary Eclipse spectrofluorimeter (Varian, Palo Alto, USA) equipped with a thermostatically controlled cuvette holder. Excitation was at either 271 or 295 nm. Emission, for both excitation wavelengths, was detected at 303, 338, and 352 nm. Fluorescence was measured at a protein concentration of 2.0 mg ml1.
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protein concentrations of 0.4 mg ml1. For protein concentration of 2.0 mg ml1, the minimal adjustable chaotrope concentration was between 0.2 and 0.5 M depending on chaotrope and fusion protein. The recorded kinetics were based on the detection of released peptide by RP-HPLC. However, proper refolding of EDDIE is a prerequisite for the autoproteolytic cleavage. Thus, at this point we prefer the term refolding and cleavage kinetics to emphasize that two different reactions are associated. It is important to point out that the respective rate constants represent apparent refolding and cleavage constants because the time course of the reactions and their interdependence is unknown and are one objective of this study, respectively. Representative refolding and cleavage time courses for EDDIE-pep6His at 0.4 and 2.0 mg ml1 are shown in Fig. 1. We observed a range of tolerable chaotrope concentrations where the ultimate cleavage yield was almost constant. The tolerable concentrations varied for the two chaotropes. For urea, the yield remained constant up to 1.4 M, whereas a significant yield reduction occurred at GuHCl concentrations higher than 0.7 M. This outcome is consistent with the report that GuHCl acts as a stronger denaturant than urea [29]. Concentrations higher than 1.2 M GuHCl led to full inhibition of autoproteolytic cleavage, whereas at urea concentration as high as 2.0 M, a yield of 30% released peptide could still be determined for the time frame investigated. The calculated rate constant k1, determined by fitting Eq. (1) to these data, decreased with increasing concentration of both chaotropic reagents. The entire data set in terms of k1 representing the influence of chaotrope concentration on cleavage for EDDIE-pep6His and EDDIE-sSNEVi-C is shown in Fig. 2. The graphs shown represent data comprising fixed protein concentrations of 0.4 and 2.0 mg ml1 with varying chaotrope concentrations (shown in Fig. 1) as well as a concentration series of 0.1–2.0 mg ml1 with chaotrope concentrations resulting from the dilution step. The decline of k1 was approximated by an exponential function in the form of: k1 ¼ k0 expðb cÞ
(2)
3. Results and discussion 3.1. Influence of chaotrope and protein concentration on refolding and cleavage of EDDIE fusion proteins The most relevant molecular characteristics of EDDIE, the fusion partners, and the respective fusion proteins are summarised in Table 2. For investigating the influence of type and concentration of chaotrope on refolding and cleavage of EDDIE fusion proteins, cleavage kinetics were determined at two concentrations for EDDIE-pep6His and EDDIE-sSNEVi-C: 0.4 and 2.0 mg ml1. These experiments were performed in the presence of increasing concentrations of urea and GuHCl in the refolding buffer. A range of 0.12–2.0 M urea and 0.06–2.0 M GuHCl final chaotrope concentration during refolding and cleavage was investigated for the fusion proteins EDDIE-pep6His and EDDIE-sSNEVi-C at
where k0 represents a hypothetical maximal rate constant, b a parameter for the decrease of the rate constant and c the chaotrope concentration. A summary of the estimated parameters is shown in Table 3. This data analysis showed that refolding and cleavage kinetics are in fact independent of protein concentration as proposed by Eq. (1) and only depend on the chaotrope concentration. As expected, this effect is more pronounced for GuHCl and represented by low values of the parameter b. Interestingly, k1 differs by one order of magnitude for the two target peptides regardless of the chaotrope system, although both represent a low molecular weight polypeptide without a distinct structure. This fact suggests dominant influence of the type of target peptide and possibly the first amino acid of the target peptide on the refolding and cleavage rate.
Table 2 Biophysical properties of peptides used for EDDIE fusion proteins. EDDIE and fusion partnersa
Amino acid sequence
pI of peptide/EDDIE fusion protein
Number of cysteines
Number of amino acids
Molecular mass (kDa)b
EDDIE pep6His K-pep6His sSNEVi-C C-sSNEVi
see Table 1 SVDKLAAALEHHHHHH KSVDKLAAALEHHHHHH KVAHPIRPKPPSATSIPAIC CKVAHPIRPKPPSATSIPAI
6.59 6.55/6.6 7.2/6.86 9.5/8.29 9.5/8.29
3 0 0 1 1
168 16 17 20 20
19.1 1.8 1.9 2.0 2.0
a
Proteins derived from codon-optimised genes are preceded by an ‘s’.
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Fig. 1. Influence of chaotrope on autoproteolysis of EDDIE-pep6His in the presence of increasing concentrations of urea (A and C) and GuHCl (B and D). Data points represent experimental values determined by HPLC analysis. Lines represent fits with Eq. (1). The protein concentration was 0.4 mg ml1 in Fig. 1A and B and 2.0 mg ml1 in Fig. 1C and D. Autoproteolysis of EDDIE fusion proteins was started by dilution of IB solutions in refolding buffer adjusting the according protein concentration. The residual chaotrope concentration represents the minimal concentration for refolding which is adjustable by dilution. Higher chaotrope concentrations were achieved by addition of chaotrope to the refolding buffer.
3.2. Influence of 1st amino acid of target peptide on refolding and cleavage of EDDIE fusion proteins
Fig. 2. Influence of chaotrope concentration on cleavage kinetics of EDDIE-pep6His and EDDIE-sSNEVi-C. Symbols represent rate constants k1 at different chaotrope and protein concentrations (0.1–2.0 mg ml1) determined by Eq. (1). Lines represent curve fits with an exponential function according to Eq. (2). Global fits were carried out for each data set comprising fixed protein concentrations of 0.4 and 2.0 mg ml1 with varying chaotrope concentrations as well as a concentration series of 0.1–2.0 mg ml1 with chaotrope concentrations resulting from the dilution step.
The refolding and cleavage kinetics of four selected EDDIE fusion proteins were investigated at four different protein concentrations ranging from 0.1 to 2.0 mg ml1 (Fig. 3): EDDIEpep6His, EDDIE-K-pep6His, EDDIE-sSNEVi-C and EDDIE-C-sSNEVi. EDDIE-pep6His and EDDIE-K-pep6His as well as EDDIE-sSNEVi-C and EDDIE-C-sSNEVi only differ in the first amino acid of the target peptides (Table 2). At protein concentrations up to 2.0 mg ml1, the yield of released peptide is independent of protein concentration and could be described properly by the proposed model for autoproteolysis of EDDIE fusion proteins defined by Eq. (1). The plots of normalised cleavage yield vs. time showed that reaction rate is strongly influenced by the type of fusion partner and the nature of its first amino acid. Insertion of Lys as the first amino acid in pep6His led to a fivefold decrease of the rate constant (3.7 104 to 7.3 105 s1) (Fig. 3A and B). Transferring Cys from the C terminus to the N terminus led to a ninefold increase of the rate constant for autoproteolytic cleavage of the EDDIE fusion proteins with the target peptide sSNEVi (2.5 105 to 2.2 104 s1) (Fig. 3C and D). Thus, for these fusion proteins, the first amino acid at the N terminus of the fusion partner has a predominant
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Table 3 Parameters calculated with Eq. (2) for chaotrope influence on autoproteolysis rate of EDDIE-pep6His and EDDIE-sSNEVi-C. k0 (s1) is a hypothetical maximal rate constant and b (M1) a parameter for the decrease of the rate constant. EDDIE-pep6His Urea 1
k0 (s ) b (M1)
(4.5 0.2) 10 1.5 0.1
EDDIE-SNEVi-C GuHCl
4
(2.2 0.4) 10 5.3 0.8
Urea 3
(4.9 0.5) 10 1.2 0.2
GuHCl 5
(8.3 1.8) 105 2.1 0.6
Fig. 3. Autoproteolysis of EDDIE fusion proteins. Kinetics of cleavage with increasing concentrations of EDDIE-pep6His (A), EDDIE-K-pep6His (B), EDDIE-sSNEVi-C (C) and EDDIE-C-sSNEVi (D) were determined by RP-HPLC analysis as described in Section 2.
influence on the reaction rate. As also demonstrated in Fig. 3, the exponential dependence of k1 on the chaotrope concentration resulted in substantially longer times for the systems with slow kinetics to reach equilibrium. Thus, it takes longer time to reach maximum yield when autoproteolysis is performed with high protein concentrations which carry higher levels of residual chaotropic reagents. In order to investigate how the refolding and cleavage are timely interconnected, we followed structural changes of the fusion proteins over time by biophysical methods. 3.3. Analysis of EDDIE secondary structure by ATR-FTIR spectroscopy By the first measurement, performed 1 min after the start of refolding, secondary structure was already evident in all cases. However, the secondary structure composition of the 4 EDDIE
fusion proteins changed with time. This is exemplarily shown for EDDIE-pep6His in Fig. 4. The maximum of the amide I band shifted to lower wavenumbers and the relative area of the peaks corresponding to b-sheets (1635–1615 cm1) increased. On the other hand the amount of a-helix decreased with time. It has been shown for several proteins that an increase of b-sheet structures, often accompanied by a loss of a-helix, correlates with aggregation [20,23]. Militello et al. attributed the peak at 1620 cm1 to intramolecular b-sheets involved in aggregation [22]. Zandomeneghi et al. also showed that a shift of the maximum to lower wavenumbers and a peak at 1620 cm1 are characteristic for intramolecular b-sheets [30]. For all EDDIE fusion proteins we observed a decrease of a-helix and concomitant increase of b-sheet structures (Table 4). The whole data set is shown in supplementary Fig. 1. Importantly, no direct link between changes in secondary structure over time and refolding and cleavage
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Fig. 4. Resolution of secondary structures in EDDIE-pep6His amide I bands at start and end point of refolding and cleavage reaction. Fourier self-deconvolution (FSD) and Gaussian curve-fitting of the amide I band was performed to resolve the respective subbands. The subband at 1618 cm1 corresponds to intermolecular b-sheets while the subband at approximately 1635 cm1 corresponds to intramolecular b-sheets.
kinetics could be observed. Therefore, the different kinetics of the four EDDIE fusion proteins cannot be attributed to structural rearrangements based on analysis of secondary structure. 3.4. Analysis of EDDIE aggregation by turbidity measurements Clearly, EDDIE formed aggregates in all cases (Fig. 5A). Absorbance started to increase immediately after establishing refolding conditions. The turbidity data was related to refolding and cleavage kinetics data (Fig. 5B). For EDDIE fusion proteins which showed fast reaction kinetics, aggregation occurred faster, Table 4 Determination of a-helix and b-sheet content of EDDIE-pep6His, EDDIE-K-pep6His, EDDIE-sSNEVi-C and EDDIE-C-sSNEVi over the time course of the refolding and cleavage process by ATR-FTIR.
Fig. 5. (A) Turbidity measurements of EDDIE fusion proteins under batch refolding conditions and (B) cleavage kinetics determined by RP-HPLC and fit to 1st order refolding model (Eq. (1)). The protein concentration for studies with EDDIE-sSNEVi-C, EDDIE-C-sSNEVi EDDIE-pep6His and EDDIE-K-pep6His was 2 mg ml1.
leading to an increase in scattered light at 350 nm. In earlier work, we stated that aggregation was not a rate limiting factor for the autoproteolytic cleavage of EDDIE fusion proteins [9]. According to turbidity data, aggregation showed the same progression as refolding and cleavage kinetics. Aggregation and release of peptide appeared to be almost parallel events, indicating that aggregation took place right after cleavage. We reasoned that EDDIE had a lower tendency to aggregate when fused to sSNEVi and pep6His variants. The relatively strong aggregation process also suggests that the increase of b-sheet content of EDDIE discussed in the ATR-FTIR section was mainly caused by the aggregation preceding formation of b-sheet structures.
Time (min)
a-Helix (%)
b-Sheet (%)
EDDIE-pep6His
1 30 1000
20.7 18.9 15.0
23.2 24.8 27.3
3.5. Analysis of EDDIE aggregation and tertiary structure by fluorescence spectroscopy
EDDIE-K-pep6His
1 60 1000
17.6 15.6 14.6
23.4 26.0 28.5
EDDIE-sSNEVi-C
1 40 1200
25.2 13.8 12.6
22.7 23.4 29.5
EDDIE-C-sSNEVi
1 60 1200
15.2 9.6 8.5
27.0 29.1 31.0
EDDIE has a known sequence of 168 amino acids [8] and is a well suited protein for fluorescence studies. It contains 9 Tyr residues, Tyr10, Tyr25, Tyr82, Tyr89, Tyr90, Tyr93, Tyr98, Tyr129, Tyr132, scattered throughout the entire sequence except the C terminus. In close proximity to the C terminus, where cleavage takes place, 2 Trp residues are located, Trp154 and Trp164 (Table 1). The sites of these residues offered the possibility to selectively gain information about changes of the whole protein structure vs. the C-terminal region. Tyr and Trp residues were both excited at 271 nm. For selective excitation of Trp residues only, we used an
Protein concentration was 2 mg ml1.
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excitation wavelength of 295 nm. The fusion partners pep6His and sSNEVi do not contain Phe, Tyr or Trp residues, and thus will not contribute to fluorescence emission. The difference of the Trp fluorescence spectrum of EDDIE between dissolved IBs (10 M urea, unfolded) and under refolding conditions was initially investigated using the EDDIE-pep6His fusion protein. Trp fluorescence is highly dependent on solvent exposure. Generally speaking, solvent exposed Trp emits at 340– 355 nm, whereas those residues buried in the interior of the folded protein fluoresce at shorter wavelengths, typically between 308 and 340 nm [31]. The emission maximum for unfolded EDDIE was determined to be at 352 nm. Under refolding conditions, the maximum shifted to 338 nm (data not shown). An emission maximum at 338 nm is close to the upper limit of 340 nm for native Trp fluorescence described in the literature [32], but is also within the limits of fluorescence for a Trp residue that is in a solvent-shielded, hydrophobic environment. Less solvent exposure results in an emission maximum shifted to shorter wavelengths. Consequently, a relatively high exposure of Trp residues to the solvent in the native state results in an emission maximum at longer wavelengths. No further significant shifts of the Trp emission maxima were observed over the extended times associated with cleavage for sSNEVi and pep6His variants. In conjunction with the results obtained from ATR-FTIR and turbidity measurements, our observations indicate that no conformational changes took place after the collapse of the protein once refolding conditions were established. To examine whether additional folding events occurred during the extended time frame of batch refolding, we investigated the ratio of emission for folded and unfolded EDDIE. Thus, we excited at 295 and 271 nm and measured emission at wavelengths 338 and 352 nm. Over the whole time range, the ratio of fluorescence emissions was constant (supplementary Fig. 2). These results indicate that neither Tyr nor Trp residues were associated with structural changes of EDDIE. Therefore, we assume that no significant structural changes occur over the observed time frame. Fluorescence intensity showed a time dependent decrease. Typical quenching data is exemplarily shown for EDDIE-sSNEVi-C in Fig. 6. Data for the other EDDIE fusion proteins is depicted in supplementary Fig. 3. This raised the question whether aggregation processes came into play. Quenching is a contact phenomenon and since no chemicals with the potential to quench were added
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during the course of refolding, the decrease of fluorescence intensity must have been caused by aggregation. The shape of the curves in Fig. 6 allowed us to draw conclusions regarding the fluorophore exposure to the solvent [32]. Quenching of fluorescence attributed to exciting Trp residues was less pronounced and intensity decreased linearly. Greater quenching of Tyr fluorescence occurred and intensity decreased rather hyperbolically. This can be explained by the fact that Trp residues were less surface accessible than Tyr residues. Trp residues were already shown to have a relatively moderate solvent exposure since native EDDIE emits at 338 nm (close to upper limit of 340 nm for native Trp fluorescence). This indicated that EDDIE was in a rather loose and flexible state. The quenching studies indicate that the C-terminal Trp residues in close vicinity to the cleavage site are more shielded from the solvent than the Tyr residues. Combining results obtained by fluorescence measurements, we propose that at the level of tertiary structure, native EDDIE is a rather unstructured protein. Importantly, the cleavage site has the highest solvent shielding which may be explained by the formation of a cavity. So far, NMR and X-ray crystallographic experiments have failed in providing a 3D-structure, which may be due to this loose conformation. Ru¨menapf et al. showed that Cys69 is essential for cleavage [6]. Our results support the findings of Ru¨menapf et al. We hypothesise that the mechanism for establishing a spatial proximity between the cleavage site and the catalytic centre of the autoprotease is the formation of a cavity comprising the folding of the C terminus towards Cys69. In summary, all applied biophysical methods did not detect significant structural changes of fusion proteins during the examined time course of autoproteolytic cleavage. Since the proper refolding of EDDIE is the prerequisite for cleavage, these results strongly indicate that refolding of the autoprotease is already completed at the first measurement. According to that, the kinetics recorded for the autoproteolytic process were largely dominated by the cleavage reaction. This is further supported by the fact, that no significant differences in secondary structure could be determined for the different fusion proteins although the reaction rates of their corresponding autoproteolytic cleavage differed up to one order of magnitude. Herein, the impact of the 1st amino acid dominated over the amino acid composition of the respective target peptide. This suggests that the cleavage rate and thus the overall kinetics of the autoproteolytic reaction are determined by the steric configuration of the cleavage site. Our investigations corroborate the model we previously developed which suggested a monomolecular reaction for autoproteolysis of EDDIE fusion proteins [9]. In fact, yield and reaction rate are independent of protein concentration. This implicates, that the initial refolding reaction preceding cleavage is also independent of the protein concentration but depends on type and concentration of chaotrope. This represents a remarkable property of EDDIE. The findings presented here allow prediction and control of preparative production of peptides using the EDDIE autoprotease fusion system. However, resolution of its atomic structure will be necessary to fully understand the cleavage mechanism. Acknowledgements
Fig. 6. Fluorescence quenching data for EDDIE-sSNEVi-C under refolding conditions. Protein fluorescence was excited at 271 and 295 nm, emission was measured at 338 nm and a protein concentration of 2 mg ml1 was used.
This work was carried out in the Austrian Centre of Biopharmaceutical Technology which is a Competence Centre within the Kind Knet programme funded by the Austrian Federal Ministry of Economics and Labour (FWF) and the provinces Vienna and Tyrol. We thank Prof. Christian Obinger and Dr. Manfred Schwanninger, Department of Chemistry at the University of Natural Resources and Applied Life Sciences Vienna, for their valuable support with respect to spectroscopic methods.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.procbio.2009.06.017.
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