Partitioning of peptide-tagged proteins in aqueous two-phase systems using hydrophobically modified micelle-forming thermoseparating polymer

Biochimica et Biophysica Acta 1601 (2002) 138 – 148 www.bba-direct.com

Partitioning of peptide-tagged proteins in aqueous two-phase systems using hydrophobically modified micelle-forming thermoseparating polymer Anna Nilsson a, Hans-Olof Johansson a, Maurice Mannesse b,1, Maarten R. Egmond b,2, Folke Tjerneld a,* a

Department of Biochemistry, Chemical Center, Lund University, P.O. Box 124, S-221 00 Lund, Sweden b Unilever Research Laboratory, Vlaardingen, The Netherlands Received 5 February 2002; received in revised form 17 July 2002; accepted 25 September 2002

Abstract Genetic engineering has been used to construct hydrophobically modified fusion proteins of cutinase from Fusarium solani pisi and tryptophan-containing peptides. The aim was to enhance the partitioning of the tagged protein in a novel aqueous two-phase system formed by only one water-soluble polymer. The system was based on a hydrophobically modified random copolymer of ethylene oxide (EO) and propylene oxide (PO) units, HM-EOPO, with myristyl groups (C14H29) at both ends. The HM-EOPO polymer is strongly self-associating and has a lower critical solution temperature (cloud point) at 12jC in water. At temperatures above the cloud point a two-phase system is formed with a water top phase and a polymer-enriched bottom phase. By adding a few percent of hydroxypropyl starch polymer, Reppal PES 200, to the system, it is possible to change the densities of the phases so the HM-EOPO-enriched phase becomes the top phase and Reppal-enriched phase is the bottom phase. Tryptophan-based peptides strongly preferred the HM-EOPO rich phase. The partitioning was increased with increasing length of the peptides. Full effect of the tag as calculated from peptide partitioning data was not found in the protein partitioning. When a short spacer was introduced between the protein and the tag the partitioning was increased, indicating a better exposure to the hydrophobic core of the polymer micelle. By adding a hydrophilic spacer between the protein and trp-tag, it was possible to increase the partitioning of cutinase 10 times compared to wild-type cutinase partitioning. By lowering the pH of the system and addition of NaCl, the partitioning of tagged protein was further increased towards the HM-EOPO phase. After isolating the HM-EOPO phase, the temperature was increased and the protein was back-extracted from the HM-EOPO phase to a fresh water phase. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Partitioning; Aqueous two-phase system; Thermoseparating polymer; Hydrophobically modified; HM-EOPO; Protein – peptide fusion

1. Introduction An aqueous two-phase system is formed when two structurally different polymers are mixed above a critical concentration in water [1]. The formed two phases are each enriched in one polymer, but the main component in both phases is water. Usually the water content is 80– 95% and, * Corresponding author. Tel.: +46-46-222-4870; fax: +46-46-222-4534. E-mail address: [email protected] (F. Tjerneld). 1 Present address: Protein Chemistry Department, Pharming Technologies B.V., Archimedesweg 4, Postbus 451, 2300 AL Leiden, The Netherlands. 2 Present address: Section of Membrane Enzymology, CBLE, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.

thus, aqueous two-phase systems constitute a mild method for separation of biomaterials. Bioseparation by using twophase systems is a fast and simple technique and is relatively easy to scale up. Proteins and other biomaterials will partition between the polymer phases depending on their properties and the system variables. Important properties of the protein for partitioning to a certain phase are size [2,3], average surface hydrophobicity [4] and net charge [5– 7]. System variables that in general are important are difference in polymer concentration and average hydrophobicity between the phases and salt composition [1,6,8]. The most commonly used two-polymer system is composed of polyethylene glycol (PEG) and dextran. Since dextran is a rather expensive polymer much research effort has been devoted on finding cost-effective alternatives [9–

1570-9639/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 7 0 - 9 6 3 9 ( 0 2 ) 0 0 4 6 2 - 4

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11]. Polymer types other than PEG are also studied. One example is the thermoseparating EOPO copolymers [3,12 – 21]. These polymers consist of randomly polymerised ethylene oxide (EO) and propylene oxide (PO) units. The EOPO copolymers display a lower critical solution temperature (LCST) in water. This phenomenon has been explained by an increase in polymer segment hydrophobicity with increasing temperature [22,23]. The thermoseparating property is strongly dependent on the hydrophilic/hydrophobic balance of the polymer with decreasing LCST for increasing PO content [24]. The thermoseparating random EOPO polymers form a macroscopic two-phase system above the LCST, where the polymer enriched phase contains more than 40% polymer and the polymer depleted phase usually less than 1% polymer [23]. For thermoseparating systems, temperature has a strong influence on partitioning due to a strong temperature effect on the polymer concentration in each phase [3]. Proteins have been found to partition almost exclusively to the water phase due to the entropic driving force, which favours partition to the water phase [3], but some hydrophobic peptides have been partitioned towards the EOPO phase [25]. In this study a hydrophobically modified random copolymer consisting of EO and PO units was used. The polymer was modified by myristyl (C14H29) groups covalently linked at each end. The HM-EOPO polymer has a cloud point of 12 jC at 3% (w/w). Above this temperature the polymer phase separates into one polymer-enriched bottom phase with 6 – 8% (w/w) HM-EOPO and one water-rich top phase with less than 1% (w/w) of polymer. Thus, due to the relatively high water concentration in the polymer rich phase, this system can be used for protein partitioning at room temperature, in contrast to simple random EOPO copolymers [26]. The HMEOPO polymer is like most hydrophobically modified polymers [27], strongly self-associating and forms micellar-like structures. The CMC is 12 AM [26]. Two different types of two-phase systems were used in this study. In one system HM-EOPO was the only polymer present, resulting in an HM-EOPO-enriched bottom phase and a water top phase (Fig. 1). However, in a separation process with cell materials, it can be more advantageous to extract the target protein in the top phase since cells, and

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other large biomaterials tend to partition towards the bottom phase due to relatively high specific gravity. By adding a few percent of hydroxypropyl starch polymer, Reppal PES 200, the phases could be inverted with the majority of the HM-EOPO collected in the top phase (Fig. 1). The target protein used in this study was the lipolytic enzyme Fusarium solani pisi cutinase, which was recombinantly expressed in Saccharomyces cerevisiae. Cutinase catalyses the hydrolysis of the water-insoluble biopolyester cutin, which covers the surface of plants [28 – 30]. However, the protein is also able to hydrolyse water-soluble esters [31]. Cutinase was genetically engineered by adding tryptophan-based tags to the C-terminal. The aim of the fusion tag was to increase the hydrophobicity of the protein and thereby direct the protein towards a more hydrophobic polymer phase. The effect of introducing a spacer between the protein and the tag was also investigated. The spacer allows the tag to extend further from the protein and thus to interact more freely with the polymer. Studies have been carried out on partitioning of fusion proteins containing tryptophan-based tags in PEG/salt [32 – 34], and EOPO/ dextran aqueous two-phase systems [20,21,35,36]. Extraction of tryptophan-tagged cutinase has been studied in systems formed by thermoseparating surfactants [37]. The presently studied phase system based on a hydrophobically modified polymer (HM-EOPO) offers the possibility to utilise the thermoseparation of the polymer for back-extraction of the tagged protein to a water phase suitable for polishing steps by chromatography. Also, we show that methods developed for prediction of the partitioning of tagged proteins [20,21,38] can be applied for predicting partitioning and tag effects in HM-EOPO-based systems. 1.1. Theoretical contributions of peptide tags to protein partitioning The fundamental driving forces for partitioning of polymeric macromolecules in aqueous two-phase systems can be understood in the framework of Flory – Huggins theory of polymer solutions [3,39]. The free energy of transfer of a macromolecule between the phases can be given as a sum over the free energy contributions of the different parts (amino acids, peptides) of the macromolecule: X DGmacromolecule ¼ ni DGi ð1Þ i

where ni denotes the number of repeating units of component i. The partition coefficient (K) is defined as the concentration of a certain substance in the top phase divided by the concentration in the bottom phase. From Eq. (1), it follows: X ln Kmacromolecule ¼ ni ln Ki ð2Þ i

Fig. 1. Schematic illustration of the aqueous two-phase systems used in the study: the one-polymer HM-EOPO/water and two-polymer HM-EOPO/ Reppal systems.

Eq. (2), however, cannot be used for native proteins since most of the amino acids are not exposed to the solution. For

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proteins with fused peptide tags that are freely exposed to the solution, the theoretical K-value of the fusion protein can be written as: ð3Þ

2. Material and methods

The free energy change for the protein partitioning when adding a tag is defined as follows:

2.1. Peptides and proteins

DGtag ¼ RT ðln Kfusion

The peptides (WP)n=(Trp-Pro)n, where n = 2 and 4, (W)4=(Trp)4 and (TGGSGG)=(Thr-Gly-Gly-Ser-Gly-Gly) were obtained from Synpep Corporation (Dublin, CA, USA). (W)n=(Trp)n, where n = 1– 3, were purchased from Sigma (St. Louis, MO, USA). (WP)=(Trp – Pro) and (P)n=(Pro)n, where n = 1, 3 and 4 were obtained from Bachem (Bubendorf, Switzerland). BSA was purchased from Sigma. The cutinase-producing S. cerevisiae MM01 strains (Mata, leu2-3, ura3, gal1: URA3, MAL-8, MAL3, SUC3) contained the expression vectors pUR7320 (encoding cutinase wild-type), pUR807 (cutinase-(WP)2), pUR806 (cutinase-(WP)4) and pUR817 (cutinase-TGGSGG-(WP)4). The synthetic cutinase gene [41] was placed behind the invertase signal sequence under the control of a GAL7 promoter. The plasmids were integrated on the chromosomal ribosomal DNA locus and the constructs contained a LEU2d gene [42], enabling selection in Leu-lacking media. The strains were constructed at Unilever Research Laboratories, Vlaardingen, NL within the EU project ‘‘Integrated bioprocess design’’, BIO4-CT96-0435. The cutinase-producing yeast strains were cultivated on agar plates in selective medium (0.67% yeast nitrogen base without amino acids, 2% glucose and 2% agar) for 48 h at 30 jC. Colonies from the plates were inoculated in 50-ml selective medium and cultivated for 48 h in 250-ml baffled shake flasks. The cultures were transferred and diluted 10fold into rich medium (2% yeast extract, 1% bactopeptone, 2% glucose and 2.5% galactose) and cultivated for 48 h at 30 jC. The culture broth was cooled and centrifuged for 10 min at 4000  g. The main protein in the supernatant was cutinase. Before partitioning, the supernatant was desalted on a PD10 column (Amersham Biosciences, Uppsala, Sweden). The molecular mass of some of the cutinase constructions has been determined by electrospray mass-spectrometry, to confirm the presence of the peptide tags (spectra not shown). For cutinase-(WP)2 the calculated molecular mass was 21175 Da and the mass-spectrometry value was 21172 Da and for cutinase-(WP)4 the calculated value was 21741 Da and the experimentally obtained value was 21739 Da. Thus, we conclude that the tags are preserved after production in S. cerevisiae.

ln Kfusion

protein

¼ ln Kwild type þ ln Ktag

obtained from peptide partitioning data is found in the fusion protein partitioning.

protein

 ln Kwildtype Þ

ð4Þ

The contribution of the tag to partitioning is here denoted as tag effect: Tag effect ¼

Kfusion protein Kwildtype

ð5Þ

The tag effect is a measurement of the preference of the fusion protein for a certain phase, when comparing with the wild-type protein partitioning. Eq. (5) is utilised when the target protein is extracted in the top phase. When the target protein is partitioned towards the bottom phase the tag effect is the inverse of Eq. (5). 1.2. Empirical formula for fusion protein partitioning The following formula has previously been used to estimate the expected partitioning value for a fusion protein [20,21]: log Kfusion

protein

¼ log Kwildtype þ log Kpeptide

ð6Þ

However, in this formula contributions from the C- and N-terminals of both the peptide and the wild-type protein are included, which will not give a correct estimate for log K of the fusion protein. In the present study log Kpeptide was replaced by (Blog K(WP)/Bn)  n, i.e. the linear regression value from the plot of log K for the (WP)n peptides versus n, multiplied by the number of WP units in the peptide tag. Thus, the contribution to log K for the fusion protein will be represented by the added residues: log Kfusion

protein

¼ log Kwildtype þ

BlogKðWPÞ n Bn

ð7Þ

1.3. Tag efficiency The tag efficiency, TE (in %), can be calculated as follows [38,40]: TE ¼

ðlog Kfusion protein  log Kwildtype Þ  100 BlogKðWPÞ n Bn

ð8Þ

The tag efficiency is a measurement of the efficiency of the tag relating to what is expected from peptide partitioning. A tag efficiency of 100% means that the expected effect

2.2. Chemicals The hydrophobically modified EOPO copolymer, HMEOPO, was a gift from Akzo Nobel (Stenungsund, Swe-

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den). The structure of the HM-EOPO polymer is shown in Fig. 2. Previously an incorrect structure of the polymer has been published [26], with ester bonds instead of the ether bonds present before the C14H29 chain in each end. An isophoronediisocyanate (IPDI) group connects the EO and PO groups, which are randomly polymerised with approximately 66 EO units and 14 PO units on each side of the IPDI group. The molecular mass of the HM-EOPO polymer is 8000 Da. Reppal PES 200, a hydroxypropyl starch polymer, constituted the bottom phase polymer in some of the systems and was obtained from Carbamyl AB (Kristianstad, Sweden). The Reppal polymer has a molecular mass of 210 000 Da. Yeast extract, bactopeptone, yeast nitrogen base without amino acids and bactoagar were purchased from Difco Laboratories (Detroit, MI, USA). All chemicals were of analytical grade.

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performed at 4 jC. The concentration of Reppal in each phase was measured by polarimetry at room temperature and all samples contained 25% (v/v) methanol to prevent clouding of the HM-EOPO polymer. The concentration of Reppal obtained from the polarimetry measurements was used to calculate the contribution of Reppal to the refractive index by using the standard curve. By subtracting this contribution from the measured refractive index, the HMEOPO concentration could be calculated. Polarimetry was measured with a polarimeter (Model AA-10) from Optical Activity (London, UK) and refractive index on a refractometer from Carl Zeiss (Oberkochen/Wu¨rtt, Germany). The tie lines within the phase diagram connect the two phases in equilibrium. The composition of the phases is found at the end points of the tie line. The volume ratio of the phases depends on where along the tie line the mixing point for the system is chosen [1].

2.3. Phase diagram 2.4. Two-phase systems A temperature – concentration phase diagram for the HMEOPO/water system was constructed by making 5-g twophase systems consisting of 3% (w/w) of HM-EOPO in water. The systems were mixed for 5 min and equilibrated for at least 20 min in a water bath at different temperatures. The systems were mixed again for 2 min and centrifuged at 800  g for 8 min at the same temperature and separated. The HM-EOPO content in the different phases was determined by refractive index by using a standard curve. The lowest clouding temperature at 3% (w/w) was determined by slowly increasing the temperature of a water bath until the polymer solution was cloudy. The system was equilibrated for at least 20 min at each temperature. To construct the HM-EOPO/Reppal phase diagram, a set of 5-g two-phase systems was made by taking appropriate amounts of the two polymer stock solutions and water. The systems were carefully mixed for 5 min and equilibrated for approximately 20 min in a water bath at 21 jC and mixed again for 2 min and centrifuged for 8 min at 800  g. The phases were separated, diluted and analysed. Standard curves for refractive index were constructed for both HMEOPO and Reppal and a standard curve for polarimetry was made for Reppal. To overcome the clouding of the HMEOPO polymer, all measurements of refractive index were

Fig. 2. The structure of the hydrophobically modified EOPO copolymer, HM-EOPO. Approximately 66 EO and 14 PO units are randomly distributed on each side of the isophoronediisocyanate (IPDI) group. A myristyl group (C14H29) is attached through an ether bond at the end of each EOPO chain.

Systems consisting of 4% (w/w) HM-EOPO and in some cases 3% (w/w) Reppal PES 200, with a total weight of 2 g, were made by weighing out appropriate amounts of a 20% (w/w) stock solution of HM-EOPO or Reppal PES 200, in calibrated test tubes. Peptides were added to the systems from pre-made stock solutions, giving a final concentration of approximately 0.1 mg peptide/g of the system mass. (W)4 and (WP)4 were dissolved in a 10% (w/w) solution of HMEOPO, due to their low solubility in water. This amount of HM-EOPO was included in the total HM-EOPO content in the phase system. The protein concentration used in the systems was 10 –15 Ag protein/g of the system mass. Sodium phosphate buffer (NaP) was used at pH 7.0 and sodium acetate (NaAc) at pH 5.3, 5.0 and 4.0. At pH 3.5 sodium citrate (Na-citrate) was used as buffer. The effect of potassium sulfate (K2SO4) as well as sodium chloride (NaCl) was investigated. Triethylammonium phosphate (Et3NHP) was used when partitioning BSA. Triethylammonium phosphate was made by titration of triethylamine with phosphoric acid to the desired pH. When no other salt was used in the system, the concentration of the buffer was 50 mM and when salt was added, the concentration of buffer was 5 mM and the salt concentration 50 mM. When sodium chloride was used as the dominating salt in the system the concentration was 200 mM and buffer 20 mM. As the concentration of the salt was 10 times higher than the buffer, the salt was the dominating electrolyte in the system [6]. The systems were mixed and equilibrated as described for the preparation of the two-polymer phase diagram. The top and bottom phases were separated and diluted for peptide respectively protein concentration determination. The partitioning of a substance is described by the partitioning coefficient, K, which is defined as the concentration of the substance in the top phase (Ct) divided by the concentration in the bottom phase (Cb). All partition coefficients are averaged values from at least duplicate experiments.

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A standard activity assay containing mixed micelles of detergent and substrate was used for concentration determination of the cutinase constructions. The substrate, p-nitrophenylbutyrate (PNPB, from Sigma), was added as a stock solution (50 mM PNPB in acetonitrile) to the assay buffer (10 mM Tris – HCl pH 8.0, 10 mM NaCl and 50 mM taurodeoxycholate, TDOC, from Sigma) to a final concentration of 1 mM. The enzymic activity was monitored spectrophotometrically at 400 nm at 21 jC by measuring the release of p-nitrophenolate. The partition coefficient was calculated as the ratio of enzyme concentration, determined by the enzymic activity, between the top and bottom phases. The enzymic activity was determined as the change in absorbance with time (dA/dt). The polymers used in the two-phase systems affected the activity of the protein. To compensate for this a blank system was prepared (all components included except protein). When the enzymic activity in the top phase was measured, a sample from the bottom phase of the blank system was included when preparing the dilution of the sample. The amount of the blank bottom phase was the same as for the dilution when the activity in the bottom phase was determined. The conditions were the opposite when the activity in the bottom phase was measured, i.e. a sample from the blank top phase was added. Thus, all samples had the same polymer concentration and the activity was equally affected by the presence of the polymers. The concentration of BSA and peptides containing tryptophan was determined by measuring the absorbance at 280 nm and subtracting the absorbance at 320 nm, as background absorbance. The contribution of the polymers was measured with blank systems (same polymer and salt composition) diluted to the same extent as the samples. Peptides lacking tryptophan were analysed at 220 nm, correcting with the absorbance at 320 nm. Methanol was added to a final concentration of 25% (v/v) in the samples to prevent clouding of the HM-EOPO polymer.

explanation of the effect is that an electrochemical potential difference is created between the two phases [1,3,6 – 8,44– 46]. The requirement for electroneutrality forces the anion and cation to partition together, but the different affinities of the ions for the two polymer phases will generate an electrochemical driving force between the phases. To investigate effects such as hydrophobicity and other non-electrostatic contributions, it is important to be able to uncouple the charge dependent effects of the partitioning. One way is to perform the partitioning at the pI of the protein, since the protein has no net charge. However, this could be a problem for many proteins, due to low solubility at this pH. A reference system, consisting of 50 mM of potassium sulfate (K2SO4), buffered with 5 mM sodium phosphate (NaP) at pH 7.0, has been used in this study. K2SO4 has earlier been shown to result in an almost zero interfacial potential in a traditional PEG/dextran system [6]. Recent studies have shown this to be valid also in an EOPO random copolymer/dextran system [21]. To investigate if K2SO4 could be used as a reference in an HM-EOPO/ Reppal system, as well as an HM-EOPO/water system, BSA was partitioned, using K2SO4 and Et3NHP as the dominating salts, respectively. In the HM-EOPO/Reppal system, with K2SO4 as the dominating salt, the partitioning was only slightly affected when varying the pH from 5 to 7 (K = 0.17 and 0.19, respectively), compared with Et3NHP (K = 0.14 and 0.44, respectively). At pH 5, BSA has a net charge of + 4 and at pH 7,  10 [47]. Similar results were obtained in the one polymer system, with K = 5.1 at pH 5 and K = 5.6 at pH 7, using K2SO4 as the dominating salt. For the systems with Et3NHP as the dominating salt the corresponding K-values were 5.9 and 3.4, respectively. The system with K2SO4 as the dominating salt can thus be used as a reference system as the charge-dependent salt effects are minimised.

3. Results and discussion 2.5. Circular dichroism (CD) measurements 3.1. Phase diagrams The CD spectra of cutinase wild-type and cutinase-(WP)4 at pH 3.5, pH 4.0 and pH 5.0, were recorded from 320 to 250 nm and 250 to 190 with a Jasco J-720 instrument (Tokyo, Japan) with cuvette lengths of 1 cm and 0.5 mm, respectively. The protein concentration was 0.5 mg/ml. The pH was adjusted with HCl. 2.6. Reference system In aqueous two-phase systems salts affect the partitioning in different ways. A salt where the anion and cation have different affinities for the two phases will influence the partitioning of a protein or peptide with a positive or negative net charge [1,6,8,43]. The mechanism for the effects of salt on partitioning in aqueous two-phase systems has been extensively investigated. One widely accepted

The temperature – concentration diagram for the HMEOPO/water system is shown in Fig. 3a. The area above the coexistence curve is the two-phase area. Polymer solutions within this area will phase separate into two macroscopic phases. Below the coexistence curve is the one-phase area. The polymer content of the HM-EOPO-enriched phase increased with temperature. Most of the partitioning was performed at 21 jC, where the HM-EOPO-enriched bottom phase has a polymer content of 7% (w/w) and the water top phase contains less than 1% (w/w) HM-EOPO. The phase diagram is qualitatively similar to corresponding diagrams for thermoseparating surfactants, e.g. C10E4 [48]. By adding a few percent of hydroxypropyl starch (Reppal PES 200) to the HM-EOPO solution, it was possible to invert the phases in the system so the HM-

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iour of HM-EOPO dominates and at higher Reppal concentration the incompatibility between the two polymers is dominating. The result of the two mechanisms for phase separation is that the binodal curve is not continuous in the phase diagram for HM-EOPO and Reppal, since phase separation occurs in the absence of Reppal. Also different from conventional two-polymer phase diagrams (see Ref. [1]) is the behaviour of the binodal curve at increased Reppal and HM-EOPO concentrations. Instead of asymptotic approach towards zero concentration of ‘‘minority’’ polymer in the phases, the curve shows increased concentration of polymers in opposite phase. 3.2. Peptide partitioning The tryptophan peptides (W)n were partitioned in both the HM-EOPO/water and the HM-EOPO/Reppal systems using the K2SO4 reference system. The preference for the HM-EOPO phase increased when the number of residues in the peptide was increased (Fig. 4a and b) in agreement with earlier results in EOPO/dextran and EOPO/water systems, where the EOPO copolymer was not hydrophobically

Fig. 3. (a) Temperature – concentration phase diagram for the HM-EOPO/ water system. The area above the coexistence curve is the two-phase area. Polymer solutions above this curve will phase separate into two macroscopic phases. Below the curve is the one phase area where no phase separation occurs. The tie lines show the composition (o) of the two phases in equilibrium. The black dot (.) represents the mixing point used in most experiments. (b) Phase diagram of HM-EOPO/Reppal PES 200/water at 21 jC. The region within the binodal curve represents the two-phase area, where a mixture of the polymers will separate into two phases. The tie lines show the composition (o) of the two phases in equilibrium. The points at zero Reppal concentration are from the temperature – concentration diagram in (a). The black dot (.) represents the mixing point used in most experiments.

EOPO phase became the top phase as illustrated in Fig. 1. The phase diagram for HM-EOPO/Reppal PES 200/water at 21 jC is shown in Fig. 3b. The concentration of HM-EOPO in the HM-EOPO-enriched phase in a system composed of 4% (w/w) HM-EOPO and 3% (w/w) Reppal was 8% (w/w), i.e. 1% more than in the one-polymer system used in most partitioning experiments. The HM-EOPO/Reppal PES 200 system differs from ordinary segregative two-phase systems such as the traditional PEG/dextran system where phase separation is induced by incompatibility between the polymers [3,49]. The phase diagram (Fig. 3b) was determined above the cloud point (12 jC) for the HM-EOPO polymer. The driving forces for phase separation are both the thermoseparating ability of the HM-EOPO polymer and the incompatibility between the HM-EOPO and Reppal polymers. At low Reppal concentration the cloud point behav-

Fig. 4. The logarithm of the partition coefficient (log K) of the peptides (W)n (5), (WP)n (o) and (P)n (4), where n = 1 – 4, as a function of the number of peptide units (n). The system contained 50 mM K2SO4 as the dominating salt and 5 mM NaP, pH 7.0. The experimental error was estimated to maximum 10% of the K-value. (a) The HM-EOPO (4%)/water system at 21 jC. Log K < 0 shows partitioning towards the HM-EOPO rich phase. (b) The HM-EOPO (4%)/Reppal PES 200 (3%) system at 21 jC. Log K>0 shows partitioning towards the HM-EOPO rich phase.

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modified [20,21]. The tryptophan peptides partitioned more strongly to the hydrophobically modified EOPO phase compared to systems containing non-modified EOPO copolymer. In the HM-EOPO/Reppal system with a tie line length of 8% (w/w) the K-value of (W)4 was above 40 (Fig. 4b), and in an EO30PO70/dextran T500 system with a tie line length of 16% (w/w) the K-value of (W)4 was 5.0 [21]. The (W)n peptides were also partitioned at pH 5.3, which corresponds to the pI of (W)2 [50], giving similar results (data not shown). The partitioning of the (WP)n peptides was practically identical to the partitioning of the tryptophan peptides, indicating that the prolines did not affect the partitioning. The proline homopeptides slightly preferred the water in respect to the Reppal phase with similar partitioning regardless the length of the peptide. The prolines were introduced in the fusion tags to prevent proteolytic cleavage of the tag during cultivation and to increase the extension of the tag by breaking a-helix structures [51]. The Blog K(WP)/Bn value, i.e. the linear regression value from the plot of log K for the (WP)n peptides versus n was  0.338 in the HM-EOPO/water system. The corresponding value in the HM-EOPO/Reppal system was 0.466. 3.3. Protein partitioning in reference system Cutinase wild-type and cutinase tagged with -(WP)2, -(WP)4 and -TGGSGG-(WP)4 were partitioned in the same systems as the peptides. The wild-type protein partitioned towards the water in respect to Reppal phase, with much stronger partitioning in the HM-EOPO/Reppal system (Table 1a and b). When introducing a tryptophan – prolinecontaining tag to the protein, the partitioning towards the HM-EOPO phase was increased. Just as in the free peptide case the partitioning increased when the length of the peptide tag was extended from (WP)2 to (WP)4. The tag Table 1 The partitioning of the cutinase constructs with 50 mM K2SO4 as the dominating salt in the system and 5 mM NaP, pH 7.0 at 21 jC Construct

K-value

Tag effect

DGtag (kJ/mol)

TE (%)

(a) Cutinase, wt (WP)2 (WP)4 TGGSGG – (WP)4

3.84 2.01 1.73 0.727

– 1.9 2.2 5.3

–  1.6  2.0  4.1

– 42 26 47

(b) Cutinase, wt (WP)2 (WP)4 TGGSGG – (WP)4

0.179 0.445 0.906 1.73

– 2.5 5.1 9.7

–  2.3  4.0  5.6

– 42 38 54

Tag effects, DGtag and tag efficiency (TE) were calculated using Eqs. (4), (5) and (8). The experimental error was estimated to maximum 10% of the K-value. (a) The HM-EOPO (4%)/water system. K < 1 shows partitioning towards the HM-EOPO rich phase. (b) The HM-EOPO (4%)/Reppal PES 200 (3%) system. K>1 shows partitioning towards the HM-EOPO rich phase.

effect (Eq. (5)) increased from 1.9 for cutinase-(WP)2 to 2.2 for cutinase-(WP)4 in the HM-EOPO/water system. In the HM-EOPO/Reppal system the tag effect was larger with values of 2.5 and 5.1, respectively, for cutinase-(WP)2 and cutinase-(WP)4. However, both the (WP)2 and the (WP)4 tagged proteins, without spacer between tag and protein, still preferred the water in respect to Reppal phase. By introducing the spacer (TGGSGG) between the protein and the (WP)4-tag the K-value was twofold increased towards the HM-EOPO phase in both the HMEOPO/water and the HM-EOPO/Reppal system. The protein was now directed from the water or Reppal phase towards the HM-EOPO phase (Table 1a and b). The partitioning of the free spacer-peptide (TGGSGG) was investigated. The partitioning coefficient was 0.89 in the HM-EOPO/Reppal system and 0.65 in the HM-EOPO/ water system. When calculating the tag efficiencies for cutinase-TGGSGG-(WP)4 (Eq. (8)) the log K-value for the free TGGSGG-peptide was added to the (Blog K(WP)/ Bn)  n value in the denominator. The highest tag efficiency was found for cutinase-TGGSGG-(WP)4. In both systems the tag efficiency was around 50% for cutinaseTGGSGG-(WP)4 (see Table 1). The free energy change for the protein partitioning when adding a fusion tag to the protein was calculated (Eq. (4)). The free energy values obtained in the HM-EOPO/water system were multiplied by  1 to be able to compare with the values obtained in the HM-EOPO/Reppal system. The DG values were all negative and decreased when increasing the length of the tag (see Table 1). A possible explanation for the relatively low efficiency of the tag in the fusion protein can be that it is not possible for the tag to interact fully with the micellar-like aggregates formed by the HM-EOPO polymer. The length of the 14˚ when fully residue tag (TGGSGG-(WP)4) is around 51 A ˚  extended (n 3.63 A, where n = 14) [51]. All end parts of the EOPO chains are presumed to be associated to myristylmicelles. The end to end distance (r) of an ideal chain with the same number of segments and segment length as the ˚ according to the EOPO-chain can be calculated to be 36 A equation [52]: r ¼ b  N 1=2

ð9Þ

The monomer length (b) of the EOPO chain is approx˚ and the degree of polymerisation (N) is 80 imately 4 A (66EO + 14PO). The length of the monomer was calculated ˚ ) and C –O (1.43 A ˚ ) bond by using the C– C (1.54 A lengths, respectively [53]. The real EOPO-chain has significantly larger length due to exclusion of volume effects from the chain. The radius of the micelle formed by the ˚ (13 C – C myristyl chains can be estimated to be 20 A ˚ ). Thus, the length of a stretched out tag is bonds of 1.54 A smaller than the radius of the micellar-like structure of the HM-EOPO polymer. In order for the tag to interact with the hydrophobic core of the micellar-like structure, it must

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be stretched out, which leads to significant loss of tagconformational entropy. Furthermore, the surface of the protein must come very close to the polymer segments surrounding the micelle core. This will lead to a decrease of the polymer chain conformational entropy, when the tag attached to the protein penetrates into the hydrophobic micelle core of HM-EOPO. The free peptides are smaller and there is no extra entropy penalty from a restrictive protein surface, thus facilitating interaction of free peptide with HM-EOPO micelles. This can explain the reduced efficiency of the peptide when attached to the protein. For the same reason the better tag efficiency for cutinaseTGGSGG-(WP)4 than cutinase-(WP)4 is due to the greater length of the spacer extended tag, which results in enhanced interaction of the hydrophobic part of the tag with the core of HM-EOPO micelles. 3.4. Salt and pH effects It has previously been shown that it is possible to affect the partitioning of proteins by adding salt in an HM-EOPO/ water system [26]. The principle is illustrated in Table 2, where the K-values for both cutinase wild-type and cutinase-(WP)4 were lowered towards the HM-EOPO-enriched bottom phase when 200 mM NaCl was included in a 4% (w/ w) HM-EOPO system, at pH 5.0. Cutinase has a pI of 7.8 and is slightly positively charged at pH 5.0 [54]. However, the wild-type cutinase still preferred the water phase while the tagged protein partitioned equally between the phases. Salt can alter the cloud point in an HM-EOPO-containing phase system and the composition of the phases can thus be changed [26]. However, the volume ratio between the phases was similar with or without the presence of the salt, indicating that the polymer composition of the phases was not dramatically altered. When the pH is lowered from pH 5.0 to pH 4.0 and pH 3.5 in systems containing 200 mM NaCl, the partitioning towards the HM-EOPO phase was increased (Fig. 5a and b) in both the HM-EOPO/water and the HM-EOPO/Reppal system. At pH 3.5 the partitioning of cutinase-(WP)4 and cutinase-TGGSGG-(WP)4 was much more increased towards the HM-EOPO phase than the wild-type cutinase in both systems. At low pH the carboxyl end group of the tag is protonated (pKa 3.5– 4.3) and becomes thus more hydrophobic [51]. This will increase the penetration of the tag into the Table 2 The effect of salt addition Construct

K-value, no salt

K-value, 200 mM NaCl

Cutinase, wt Cutinase-(WP)4

6.08 1.91

2.47 1.03

Partition coefficients of cutinase wild-type and cutinase-(WP)4 in the HMEOPO (4%)/water system at 21 jC with 20 mM NaAc, pH 5.0; 200 mM NaCl was included in the salt containing systems. The experimental error was estimated to maximum 10% of the K-value. K < 1 shows partitioning towards the HM-EOPO rich phase.

Fig. 5. The logarithm of the partition coefficient for the cutinase constructions versus pH. The systems contained 200 mM NaCl and 20 mM buffer. At pH 5.0 and 4.0 the buffer was NaAc and at pH 3.5 Nacitrate. The experimental error was estimated to maximum 10% of the Kvalue. Cutinase, wild-type (.), cutinase-(WP)4 (n) and cutinase-TGGSGG(WP)4 (E). (a) The HM-EOPO (4%)/water system at 21 jC. Log K < 0 shows partitioning towards the HM-EOPO rich phase. (b) The HM-EOPO (4%)/Reppal PES 200 (3%) system at 21 jC. Log K>0 shows partitioning towards the HM-EOPO rich phase.

hydrophobic core of the micelle compared to the situation when the end group is charged. This could explain the larger increase of the partitioning of cutinase-(WP)4 and cutinase-TGGSGG-(WP)4 towards the HM-EOPO phase compared to the wild-type, at pH 3.5 – 4. Another possible explanation could be that the protein starts to unfold. To evaluate if the effect at pH 3.5 was caused by conformational changes of the protein, CD-spectra of cutinase wildtype and cutinase-(WP)4 were recorded (spectra not shown). Since the HM-EOPO polymer disturbed the spectra, CD was measured with only protein present in the samples. The spectra for both cutinase wild-type and cutinase-(WP)4 were not altered neither in the far nor the near UV region when the pH was lowered from pH 5.0 to pH 3.5. Furthermore, the protein showed full activity after the partitioning at these pH values. 3.5. Temperature effect The effect of temperature on the partitioning in the HMEOPO/water system was investigated, both for peptide and

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protein partitioning, with (W)2 respectively cutinase wildtype and cutinase-(WP)4. As can be observed in the concentration-temperature diagram for the HM-EOPO/water system (see Fig. 3a), the polymer concentration in the polymer-rich phase was increased when increasing the temperature. In the (W)2 case, the partitioning towards the HM-EOPO phase was increased when the temperature was raised. A van’t Hoff graph of ln K against T -1 for (W)2 at 17 jC (290.15 K) to 29 jC (302.15 K) gave a linear correlation (Fig. 6). Since the slope is  DH/R, the average enthalpy of transfer of the peptide from the polymer-enriched phase to the water phase was calculated to be DHtransf =  17.5 kJ/ mol. The intercept of the van’t Hoff graph is DS/R and the average entropic part of the free energy of transfer was calculated to be  TaverageDStransf = 20.0 kJ/mol. The free energy change of transferring the peptide from the polymer to the water phase is larger than zero (DG = 2.5 kJ/mol), and thus partitioning of the peptide towards the HM-EOPO phase is favoured. The partial molar combinatorial entropy of the peptide is lower in the HM-EOPO phase due to the lower number density of molecules in this phase [3]. Thus, partitioning to the water phase is entropically favoured. One explanation for the peptide still preferring the HM-EOPO phase (Fig. 4a) could be the loss of entropy in the water phase due to the water-cage formation around the peptide, i.e. the ‘‘hydrophobic effect’’ [55,56]. Thus, hydrophobic interaction of peptides with polymer micelles favours partition to HMEOPO phase. The van’t Hoff graph in Fig. 6 gives only an average DHtransf of the transferring of the peptide from the polymer phase to the water phase. This is because the phase diagram changes relatively strongly with temperature since the polymer concentration in the HM-EOPO phase increases with the temperature. Thus, the calculated transfer enthalpy encapsulates both the enthalpy of transferring the peptide from the polymeric phase to the water phase and the change in interaction when the polymeric phase becomes more concentrated.

Fig. 6. A van’t Hoff plot of ln K for the dipeptide (W)2 versus 1/T in the HM-EOPO/water system. The systems contained 50 mM NaAc, pH 5.3. The experimental error was estimated to maximum 10% of the K-value. Ln K < 0 shows partitioning towards the HM-EOPO rich phase.

Table 3 The effect of temperature on cutinase wild-type and cutinase-(WP)4 partitioning in the HM-EOPO (4%)/water system at 21 jC Temperature (jC)

K-value, cutinase, wt

K-value, cutinase-(WP)4 (tag effect)

21 26

2.47 8.08

1.03 (2.4) 2.41 (3.4)

In each system 200 mM NaCl and 20 mM NaAc, pH 5.0, were included. The experimental error was estimated to maximum 10% of the K-value. K < 1 shows partitioning towards the HM-EOPO rich phase.

The partitioning of cutinase and cutinase-(WP)4 at two different temperatures, 21 and 26 jC respectively, is shown in Table 3. The tag effect increased as expected from the peptide partitioning data. However, both the wild-type and the fusion protein preferred the water phase, and the partitioning towards the water phase increased at higher temperature. The larger affinity of the tag towards the HMEOPO phase was not enough to overcome the exclusion of the protein from the HM-EOPO phase. 3.6. Effect of polymer concentration The effect of polymer concentration on partitioning of cutinase-(WP)4 was investigated (Table 4) in the HMEOPO/Reppal system. The highest K-value 1.72, indicating a preference for the HM-EOPO phase, was obtained in the system with the lowest polymer content. At higher polymer concentrations the protein was partitioned towards the Reppal enriched phase. Generally, the phase with highest polymer concentration is entropically least favourable (in terms of combinatorial molar entropy). Thus, considering only the entropic driving force, a substance will be partitioned to the phase with lower polymer concentration [3], i.e. the difference in total polymer concentration between the phases corresponds to an entropic driving force. This entropic driving force is largest in the system with lowest total polymer concentration (Table 4). The HM-EOPO rich phase is the entropically least favourable for partitioning, as the polymer concentration is larger in the HM-EOPO-rich phase than in the Reppal-rich phase (Table 4). However, cutinase-(WP)4 was partitioned towards the HM-EOPO-rich phase at low polymer concentration. Thus, the dominating driving force for the partitioning of the fusion protein should be of enthalpic nature, and can be explained by an effective attraction to the HM-EOPO phase due to a hydrophobic effect of the peptide tag. At higher polymer concentrations in the system, the difference in total polymer concentration between the phases diminishes (system 3 in Table 4), which would increase the driving force for partitioning to the HMEOPO phase, due to the affinity of the peptide tag for this phase, i.e. an increased K-value is expected. The observed decrease of the K-value indicates that the change in polymer composition is relatively more important than the difference in polymer concentration. This can be understood by the amphiphilic character of cutinase-(WP)4. The wild-type

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Table 4 The effect of polymer concentration on cutinase-(WP)4 partitioning in the HM-EOPO/Reppal system at 21 jC System 1 System 2 System 3

%HM-EOPO

%Reppal

%HM-EOPOtop

%HM-EOPObot

%Polymertop

%Polymerbot

K-value

4.0 5.0 6.0

3.0 4.0 5.0

8.3 8.9 10

1.2 1.6 1.9

9.2 10 11

5.6 8.1 10

1.72 0.895 0.327

In each system (1, 2 and 3) 200 mM NaCl and 20 mM NaAc, pH 4.0, were included. The experimental error was estimated to maximum 10% of the K-value. K>1 shows partitioning to the upper HM-EOPO rich phase. The estimated %HM-EOPO (in (w/w)) in respective phase is calculated using the phase diagram shown in Fig. 3b. The %polymer in the top and bottom phases means the total polymer concentration in each phase.

protein prefers the more hydrophilic Reppal phase at all pH values; see Fig. 5b. The addition of a hydrophobic tag makes the fusion protein amphiphilic. Due to amphiphilic properties, the fusion protein has attractive interactions to both Reppal and HM-EOPO polymers, where the interaction with Reppal becomes dominating at higher polymer concentrations. 3.7. Back extraction One way to take advantage of the exclusion of protein from the HM-EOPO phase observed with increasing temperature and polymer concentration is to introduce backextraction in a second step in a purification procedure. In the first step the target protein is partitioned to the HM-EOPO polymer phase. The HM-EOPO-rich phase is isolated in a separate container and a fresh water phase is added, i.e. an HM-EOPO/water system is formed. When the temperature is increased the protein will be partitioned to the water phase, as shown in Table 3. The protein can thus be collected in a water phase free of polymers. The principle was demonstrated with cutinase-TGGSGG-(WP)4. After partitioning the protein in an HM-EOPO/Reppal system at pH 5.0 with 200 mM NaCl included in the system at 21 jC (K = 2.5, i.e. partitioning towards the HM-EOPO phase), 1 g of the top phase was added to 1 g of water. The system was mixed and placed in a water bath at 45 jC. The second partitioning resulted in a K-value of 3.4, i.e. the protein partitioned towards the (upper) water phase.

polymer aggregates to a larger extent than when the tag is attached to the protein. By adding a hydrophilic spacer between protein and trp-tag it was possible to increase the partitioning of cutinase 10 times compared to wild-type cutinase partitioning. By adding salt to the system and decreasing the pH, the partitioning could be further increased. After the partitioning of the protein towards the HM-EOPO phase, the protein could be back-extracted to a fresh water phase by increasing the temperature. By addition of a few percent starch polymer (Reppal) the phases could be inverted due to changed densities of the phases. Thus, by selecting the phase system it is possible to choose whether the target protein should be extracted in the top or bottom phase, which in each case would be the HMEOPO-rich phase. The Reppal content (3% (w/w)) is much less than what is needed in a traditional two-polymer system. The results show that hydrophobic peptide tags can enhance partitioning of the target protein to a thermoseparating polymer phase. With a micelle-forming polymer, it is possible to utilise a one-polymer aqueous phase system where the target protein can be back-extracted into a water phase. Thus, extraction of hydrophobically tagged proteins with aqueous phase systems based on HM-EOPO opens possibilities of performing effective isolation of recombinant proteins with lower chemical costs in biotechnological processes.

Acknowledgements 4. Conclusions Hydrophobically modified polymers can be used for partitioning of recombinant proteins tagged with tryptophan residues. By adding tryptophan-based fusion tags to cutinase, the partitioning towards the HM-EOPO phase was increased. The partitioning of the fusion proteins could be qualitatively determined from peptide partitioning data. The longer the tag the larger is the increase of the partitioning, just as expected from peptide partitioning. Prediction of the tagged protein partitioning was poor due to decreased tag efficiency of the peptide tags in the fusion protein. The low efficiency of the tag could be due to the lower accessibility of the hydrophobic core of the HM-EOPO micellar-like structure. The peptides can move more freely in the solution and interact with the

The financial support by the EU Project BIO4-CT960435 is gratefully acknowledged. The HM-EOPO polymer was kindly provided by Leif Karlsson, Akzo Nobel (Stenungsund, Sweden).

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