Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His6 tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L−1 could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process.
Introduction
Since more than 15 years, the interest in biodegradable plastics (polymeric materials which can be cleaved by enzymes and assimilated by microorganisms, respectively) is steadily raising and nowadays a number of commercial products are on the market. Polymers containing ester groups, especially polyesters, play a central role as they are easily subject to microbial attack, the degradation rate being largely dependent on their physical structure and chemical composition. First attempts to produce biodegradable plastics were based on natural polymers like poly(β-hydroxybutyrate) (PHB). However, their material properties proved to be insufficient and could not compete with those of synthetic polyesters. Tokiwa and Suzuki [1] already showed that also synthetic aliphatic polyesters are accessible to a biological degradation. However, aliphatic polyesters exhibit similar disadvantages concerning their material properties (e.g. minor tensile strength, low melting points) and therefore are hardly applicable for every day life products. On the other hand, polyesters such as poly(ethylene-terephthalate) (PET) and poly(butylene-terephthalate) (PBT) exhibit excellent material properties, but were classified as non-biodegradable. Therefore, aliphatic–aromatic copolyesters, which combine both acceptable use properties and sufficient biodegradability [2] were developed. The material properties desired for specific applications can be easily adjusted by the ratio of the aliphatic to the aromatic diacid [3, 4]. Commercial products are available now under the trade name Ecoflex® (BASF AG, Germany).
Recently, a thermophilic actinomycete was isolated and identified as Thermobifida fusca (former name: Thermomonospora fusca) exhibiting a remarkable degradation capability for aliphatic–aromatic copolyesters [5, 6]. The extracellular hydrolase (TfH) responsible for the degradation of these copolyesters was purified from the culture broth and classified as a serine hydrolase with the highly conserved G-H-S-M-G motif [7]. The mature hydrolase consists of 261 amino acids, corresponding to a molecular weight of 28.2 kDa. Maximum activity was found at 65–70°C. The extracellular hydrolase gene was located on an operon containing two nearly identical genes (bta1 and bta2) with an amino acid identity of 92% [6].
The enzyme is only expressed by T. fusca if a polyester, such as poly(1,4-butandiole adipate-co-1,4-butandiole terephthalate) (BTA) or poly (ε-caprolactone) (PCL) is present in the medium. Gouda et al. [8] observed, that the amount of enzyme excreted in the medium correlates with the amount of polyester added to the culture. This induction behaviour as well as adsorption and inhibition phenomena [8] complicate a large-scale production of this enzyme (TfH) and therefore, a simpler to handle recombinant expression system for producing TfH, independent of the presence of the polymer was highly desirable, particularly, as recent investigations showed that TfH is capable of degrading even PET [9].
In this paper, we report on the heterologous expression of the polyester-degrading hydrolase from T. fusca in E. coli TG1. To this end, the TfH-encoding gene (bta1) was cloned together with a C-terminal His6-affinity tag and the ompA-secretion signal under the control of a thermo-inducible promoter. This strategy should enable translocation of the gene product by the Sec-transport system from the cytoplasm into the periplasmic space, where the properly folded TfH should accumulate after cleavage of the OmpA sequence. Several cultivation strategies and purification routes were considered. The aim of the investigations was to provide sufficient amounts of TfH for larger-scale polyester degradation studies (low purification grade) and a highly purified TfH for mechanistic studies and structural analysis.
Materials and methods
Cloning of the recombinant His6-tagged TfH
Escherichia coli TG1 (DSM 6056) was used for the production of the recombinant hydrolase from Thermobifida fusca (rTfH). TG1 is characterized by the following genetic markers: supE hsdΔ5 thiΔ(lac-proAB) F´[traD36 proAB+ lacIq lacZΔM15] [10].
The vector pCytexP1-OmpA-bta1 [6] was digested with NdeI and EcoRI to obtain a DNA fragment consisting of 876 bp. This DNA fragment was used as a template to clone a His6 tag at the C-terminal end using the primer set bta1-rev (5′-TAC TTA GGA TCC TTA TCA GTG GTG GTG GTG GTG GTG CTC GAG GAA CGG GCA GGT GGA GCG-3′) and bta1-for (5′-GTC ATG GGC CAC TCC ATG GGC-3′). The DNA fragment was amplified using VENT DNA polymerase (New England Biolabs, USA), the cycle programme was run at 95°C for 2 min, 30 cycles of 30 s at 95°C, 30 s at 50°C and 2 min at 55°C, followed by an extension reaction for 5 min at 55°C. The reaction mixture was designed according to the description of the manufacturer. The resulting PCR-fragment rbta1–His6 was purified using the JETQUICK PCR Fragment purification kit (GENOMED GmbH, Germany). rbta1–His6 was ligated to a DNA fragment obtained from the digestion of the origin vector with NcoI and BamHI. The resulting plasmid pCYTEXP1-OmpA-bta1-His6 with the secretion signal of OmpA, the hydrolase from T. fusca (TfH) and the affinity tag was used for the expression of rTfH.
Transformation of pCYTEXP1-OmpA-bta1-His6 into E. coli TG1 was done by electroporation. Expression of the recombinant TfH (rTfH) was controlled by the strong tandem promoter λpRpL, which is expressed by the thermolabile repressor cI857.
Media
Louria-Bertani medium (LB medium): 10 g L-1 tryptone, 10 g L−1 NaCl 5 g L−1, yeast extract and 0.8 g L−1 glucose was used for cultivations of E. coli TG1 in the complex medium [11]. The medium was autoclaved at 121°C for 20 min. Before cultivation, ampicillin (100 μg mL−1) was added.
A defined medium as described by Korz et al. [12] was used for batch and fed-batch cultivation of E. coli TG1. The medium contained (per litre): (NH4)2PO4 4 g, KH2PO4 13.3 g, citric acid 1.7 g, glucose 25 g, MgSO4 1.2 g, trace elements (Fe(III)citrate·H2O 100 mg, CoCl2·6H2O 2.5 mg, MnCl2·4H2O 15 mg, CuCl2·2H2O 1.5 mg, H3BO3 3 mg, Na2MoO4·2H2O 2.5 mg, Zn(CH3COO)2·2H2O 13 mg, EDTA 8.4 mg) and 100 μL antifoam (Sigma Antifoam 204, Sigma-Aldrich, USA). MgSO4 and glucose solution were autoclaved separately from the phosphate buffer and trace elements at 121°C for 20 min. pH was adjusted to 6.8 with 5 M NaOH, before. Ampicillin 100 mg L−1 and thiamine hydrochloride 4.5 mg L−1 were sterilized by filtration (Sterivex, cut off of 0.2 μm, Millipore, USA) and added before starting the cultivation.
The feed solution for fed-batch cultivations contained the following components (in 1 L demineralized water): glucose 300 g, MgSO4 7.6 g, trace elements (Fe(III)citrate·H2O 16.2 mg, CoCl2·6H2O 1.5 mg, MnCl2·4H2O 8.9 mg, CuCl2·2H2O 0.9 mg, H3BO3 1.9 mg, Na2MoO4·2H2O 1.5 mg, Zn(CH3COO)2·2H2O 6.1 mg, EDTA 4.9 mg), thiamine hydrochloride 4.5 mg and ampicillin 100 mg. The feed solution was sterilized by filtration.
Cultivation
A bioreactor (Biostat B2 , B. Braun, Melsungen, Germany) with 2 L working volume connected to an exhaust gas analysis unit (S710, Sick Maihak, Germany) was used for the cultivations. Bioreactor control was done by a computer, operating the MFCS software (B. Braun). Feed and base reservoirs were stored on scales connected to the computer. Starting conditions of the reactor were set to 30°C, 500 rpm, pH 6.8 and 1 L min−1 airflow. pH was controlled by adding either 25% NH3 (in case of the complex medium: 5 M NaOH) or 1 M H3PO4. The minimum level of dissolved oxygen (pO2) was set to 20% and was adjusted automatically by the stirrer speed and aeration rate. If the stirrer speed and aeration rate run at maximum, pure O2 had to be enriched to the inlet airflow. Antifoam was added manually to the cultivation under sterile conditions, if necessary.
Pre-cultures were carried out in the respective medium of the main culture by inoculation with a glycerol stock of E. coli TG1 containing the plasmid pCYTEXP1-OmpA-bta1-His6 at 30°C and 180 rpm for 12 h. Batch and fed-batch cultivation in the bioreactor was started by adding 20 mL of preculture.
In batch cultivation, induction of rTfH production was started at an OD of 0.5–0.6 by raising the cultivation temperature from 30 to 39°C or 42°C, respectively, within about 15 min.
In the fed-batch cultivation, feed was started after the initial glucose was consumed as indicated by a sharp increase in pO2. An exponential feed profile was used. For a desired specific growth rate μset (h−1), the volumetric feed rate Fglc (L h−1) was calculated from (Eq. 1) [13]:  where (XV)0 is the biomass (g) at feed start, tf the time (h) after feed start, YX/S the biomass yield coefficient (0.4 g g−1) and S the glucose concentration in the feed (g L−1). Substrate consumption by maintenance is not considered in Eq. 1. Therefore, the actual growth rate may be less than the set value (μset = 0.12 h−1). Feed rate and volume variations were corrected automatically by the MFCS software. The expression of rTfH was initiated at a biomass level of about 40 g L−1 by shifting the temperature to 39°C, which was found optimal in the preliminary runs. After induction, the feed-rate profiles were kept constant at the value of the induction time or it was continously increased as given by Eq. 1 for μset = 0.12 h−1.
Analytical methods
Cell-mass concentration was followed by OD at 600 nm (Ultrospec 3100 pro, Amersham Biosciences, Sweden). For the determination of the dry cell weight (DCW), 1 mL of the culture was added in triplicate to pre-weighed tubes and centrifuged for 5 min at 13,000g and 4°C. After a subsequent washing step, the pellet was dried until constant mass at 45°C under vacuum.
Extracellular glucose and metabolites such as acetate, formate, succinate, propionate and isobutyrate were determined in the supernatant. To this end, the supernatant was heated up to 80°C for 5 min and centrifuged (13,000g, 10 min, 4°C) to eliminate the precipitated proteins. The supernatant was additionally filtered (pore size 0.2 μm) before applied into the HPLC (Shimadzu, Japan) using an Aminex HPX-87H column (Biorad, USA). 5 mM H2SO4 with a flow rate of 0.6 mL min−1 was used as mobile phase at a temperature of 60°C.
Electrophoresis
SDS-PAGE was performed with a Mini Protean® 3 apparatus (Bio-Rad, USA) according to the manufacturer’s recommendations. The samples were run on 12% polyacrylamide gel. Molecular mass marker was purchased from Bio-Rad. Proteins were stained with Comassie-Blue [11] or silver [14].
Proteins were transferred onto a 0.45-μm PVDF membrane (Immobilon™-P Transfer Membrane, USA) using a Semi-Dry Transfer Unit (Semiphor™ Hoefer TE® 70 Series, Amersham Bioscience, Sweden). rTfH was detected by Comassie-Blue staining.
Hydrolase activity
Hydrolase activity was measured by monitoring the cleavage of p-nitrophenylpalmitate (pNPP) as described by Schmidt-Dannert et al. [15]. The released p-nitrophenol was determined spectrophotometrically (Ultrospec 3100 pro, Amersham Biosciences, Sweden). pNPP solution was freshly prepared by mixing 1 mL pNPP dissolved in 2-propanol at a concentration of 3 g L−1 with 9 mL 20 mM phosphate buffer (pH 7.1, sodium taurocholate 2.3 g L−1, gum arabic 1.1 g L−1). Immediately after addition of the enzyme sample the absorption was measured at 400 nm and 25°C against a sample without enzyme for 2 min. One unit was defined as the amount of enzyme that caused the release of 1 μmol p-nitrophenol per minute under test conditions. The extinction coefficient of p-nitrophenol is 9.62 cm2 μmol−1. To compare the photometrically determined pNPP activity units with a previously used activity test [8] the cleavage of the polyester bonds of PCL (poly(ε-caprolactone)) films in a pHstat titration system was additionally measured as described by Gouda et al. [8]. It was found that one pNPP enzyme unit corresponds to three PCL units.
Localization of rTfH
In this paper, it is discriminated among the various rTfH fractions located in the extracellular medium, the periplasmic space and the cytoplasm as soluble or insoluble protein. The extracellular fraction (EF) was obtained from the culture supernatant after centrifugation (13,000g, 5 min, 4°C). A variation of the protocol of Neu et al. [16] was used to obtain the periplasmic fraction (PF). Biomass (about 37 mg) corresponding to ten OD was resuspended in 1 mL ice-cold 30 mM Tris–HCl (pH 8.0) containing 20% sucrose and 1 mM EDTA and after incubation for 10 min at room temperature centrifuged at 13,000g and 4°C for 10 min. The supernatant was separated from the cell pellet and stored at an ice bath. The cell pellet was again resuspended in 1 mL ice-cold 5 mM MgSO4 solution and was slightly rotated on ice for 10 min. After centrifugation the supernatant was pooled with the previously obtained supernatant giving the total periplasmic proteins (PF). Each fraction (EF and PF) was stored at −20°C until further use.
Soluble proteins harboured in the cytoplasm were obtained by resuspending the cells in 275 μL PBS buffer [11] [pH 7.2, 10% (v/v) BugBuster, 0.1% (v/v) benzonase (both Novagen, USA), 275 U lysozyme], slightly rotated at room temperature for 20 min and centrifuged at 13,000g and 4°C for 25 min. The supernatant formed the soluble cytoplasmic fraction (sCF). To gain the insoluble cytoplasmic protein fraction (iCF) cells were again resuspended in PBS buffer (10% (v/v) BugBuster, 27.5 U lysozyme) and after incubation for 5 min diluted sixfold with PBS buffer (1% (v/v) BugBuster). After centrifugation at 5,000g and 4°C for 15 min cells were washed twice with 800 μL PBS (1% (v/v) BugBuster). Final centrifugation was done at 13,000g and 4°C for 25 min. The pellet was then resuspended in 275 μL of 1% (w/v) SDS.
Purification of rTfH
To gain a rTfH solution of low purification grade 75 mg biomass was resuspended in 2 mL ice-cold PBS buffer, pH 7.2 [11]. Cells were disrupted by ultrasonification (Sonopuls HD 2070, Sonotrode UW 2070, Bandelin electronics, Germany) in an ice bath using three intervals each of 30 s pulse and 2 min break. Subsequently, disrupted cell were denaturated by heat at 55°C for 10 min, and centrifuged (13,000g, 25 min, 4°C). The supernatant was stored at −20°C.
Highly purified rTfH was recovered from the extracellular and the PF (see Localization of rTfH section). Chelating Sepharose™ Fast Flow Gel (Amersham Bioscience) was activated as described by the manufacturer and added to the solution harbouring rTfH (1 mL chromatographic material was used for 400 U). The suspension was stirred for 1 h and filled then into a XK 26/60-column (Amersham-Bioscience) and after the chromatographic material has settled, the column was connected to the FPLC system (LCC-500 Plus, Amersham Bioscience, Sweden). Column was washed with 3 column volumes (CV) binding buffer containing 20 mM Tris (pH 7.5), 500 mM NaCl and 10 mM imidazole at a flow rate of 4 mL min−1. Elution of the bound rTfH was done by a linear increase of the imidazole concentration to 500 mM within 3 CV. Fractions with activity were pooled and concentrated by ultrafiltration (Amicon Stirred Cell 1050 or 1010, Amicon, USA) using a regenerated cellulose membrane (YM Ultrafiltration Membranes, cut off: 10 kDa, Amicon, USA). 6±0.5 mL of the concentrated enzyme was applied manually to the gel filtration column (HiLoad 26/60 Superdex 75 prep grade, Amersham Bioscience) equilibrated with 0.9% NaCl. The flow rate was set to 2 mL min−1 to elute the proteins. Fractions with activity were pooled and stored at −20°C until further use. Whenever necessary, rTfH solution was concentrated by ultrafiltration.
Protein determination
Protein was determined by Roti®-Nanoquant (Roth, Germany) as described by the manufacturer.
Results
Batch cultivations
E. coli TG1 harbouring the plasmid pCYTEXP1-OmpA-bta1-His6 was firstly cultivated batchwise with complex medium in 1 L scale monitoring OD, DCW, glucose and metabolites in the supernatant. At an OD of about 0.6, cultivation temperature was raised to either 39 or 42°C to induce rTfH production. After starting induction, generation of rTfH was pursued by the spectrophotometric assay in the extracellular and PFs. Results are shown in Fig. 1.
Fig. 1Expression of rTfH in E. coli TG1 during batch cultivation with LB medium. Cells grew at 30°C to an OD of 0.6 before starting induction by a temperature shift to 39 or 42°C, a growth and CO2 generation, b rTfH activity in different locations, extracellular fraction (EF; open square), periplasmic fraction (PF; open circle), sum of all fractions (continuous line), c glucose and acetate in broth
Glucose present in the complex medium is quickly consumed at about 1.4–2 h after induction (Fig. 1c). Consumption of glucose is accompanied by formation of acetate, its extracellular concentration peaks at 39°C at 2 h (3 h at 42°C, respectively), which is remetabolized after total glucose consumption. The consumption pattern is reflected by biomass development and CO2 generation (Fig. 1a) which show a two- and three-phase behaviour, respectively. Firstly growth rate is highest as long as glucose is available (induction at 39°C). The following consumption of the intermediate acetate leads to reduced growth rate. After acetate consumption the growth rate further declines and finally growth ceases, however, CO2 evolution is yet observed in the stationary phase. From the biomass and CO2 evolution, one has to conclude that in all cultivation phases considerable amounts of constituents of the complex fractions of the medium are also consumed. For instance, the 1.5 g L−1 biomass (2 h after induction at 39°C) cannot be generated alone by glucose consumption. Unfortunately, the complex constituents are not known. Therefore, a carbon balance is not possible.
The production of the hydrolase rTfH is shown in Fig. 1b. Both induction profiles, shifting the temperature to 39  or 42°C, revealed similar rTfH production kinetics. Shortly after induction rTfH is only found in the periplasm. However, in the course of the cultivation, rTfH did not accumulate in the PF as expected due to the leader signal OmpA. Surprisingly rTFH was entirely found in the extracellular medium at the end of the cultivation. At an induction of 42°C the promoter λpRpL in front of the bta1 gene is stronger than at 39°C, but expression of rTfH is nearly finished about 6 h after induction resulting in a lower volumetric activity (only 60%). About 4 h after induction, a remarkable increase of extracellular rTfH activity was observed which obviously correlates with a high metabolic activity as indicated by the second strong increase in CO2 evolution. The rise in rTfH activity cannot solely originate from translocation but suggest additional secretion from cytoplasm. Apparently expression of rTfH is faster than its transport through the cytoplasmic membrane in the early phase shortly after induction. In shake-flask cultivation, the rTfH production was monitored in the cytoplasm as soluble and insoluble cytoplasmic fractions (iCFs; data not shown). Maximum hydrolase activity was achieved after about 7.5 h summing up all compartments. Subsequently, the total hydrolase activity was slightly decreased, and finally rTfH was only detected in the supernatant.
To study the influence of medium composition on recombinant TfH production and to follow the transport of the enzyme into the medium, batch cultivations with the defined medium containing 23 g L−1 glucose were carried out (Fig. 2). rTfH was again induced at low OD. The course of the cultivation with the defined medium is remarkably different from that using the complex medium. Raising the temperature to either 39°C or 42°C caused firstly a low growth rate. The consumption of glucose was coupled with acetate formation; 8 h after induction at 39°C, growth rate rised to about 0.5 h−1 (0.6 h−1 at an induction at 42°C) and both glucose and acetate were consumed while a low amount of succinate was generated which was remetabolized after exhaustion of other C-substrates. As expected, growth was directly coupled with CO2 evolution. The respiration coefficient RQ was around 1, the yield coefficient (biomass on glucose) was determined as 0.4 g g−1 for the batch cultivation at 39°C. Over the entire induction period (about 23 h), carbon balances of the cultivation with the defined medium were close to  100 ± 3%.
Fig. 2Expression of rTfH in E. coli TG1 during batch cultivation with the defined medium. Cells grew at 30°C up to an OD of 0.6 before starting induction by a temperature shift to 39°C or 42°C, a growth and CO2 generation, b rTfH activity in different locations, EF (open square), PF (open circle), sum of all fractions (continuous line), c glucose and metabolites in broth
In contrast to the cultivation in LB medium, succinate was detected in the extracellular medium under both the induction profiles. Succinate is a typical product of the anaerobic metabolism of E. coli. It is produced during the mixed acid fermentation and the fumarate reduction. Both pathways are strictly regulated by Fnr the global regulator of the anaerobic metabolism [17]. With the applied experimental approach, anaerobiosis can be excluded. The formation of succinate was coupled with the CO2 evolution and hence with a high metabolic activity. This may lead to an imbalance in the citric cycle or reduction equivalents. Unfortunately, other metabolites of the citric cycle were not determined in the supernatant supporting this assumption. Similar observations were made by Korz in batch cultivations using the same defined medium. He correlated the succinate formation with the amount of CO2 raising in the exhaust gas [18].
While secretion of rTfH in the periplasm started immediately after induction, the rate of rTfH release in the cultivation medium correlates with the growth rate, i.e., growth and product transport from the cytosol via the periplasm into the extracellular space are coupled. At the end of the cultivation, almost the entire product was located outside the cells. The final results of the batch cultivations at controlled pH with the complex and the defined medium are summarized in Table 1. For induction in both media, by shifting the temperature to 39°C gives better results than the increase to 42°C. While the volumetric activities achieved for the complex and the defined media at the same induction temperature are comparable, the specific activities differ significantly due to the higher biomass obtained with the defined medium. As the induction time is considerably longer for the cultivation in the defined medium, the specific productivities differ even more. These results clearly indicate that production of the rTfH in batch cultivation becomes more attractive when the complex medium is employed.
Table 1Summary of results on rTfH production in batch cultivationsComplex mediaDefined mediaInduction temperature(°C)39423942Biomass (gDCW L−1)2.22.19.89.6Induction time (h)9.962323.7Volumetric activity (103 U L−1)8.084.437.52.84Specific activity (103 U gDCW−1)3.672.110.770.30Volumetric productivity [103 U (L h)−1]0.820.740.330.12Specific productivity [103 U (gDCW h)−1]0.3730.3520.0340.013
During the induction phase, the protein content in the culture supernatant was analysed by SDS-PAGE (Fig. 3). About 4 h after the temperature upshift, the first protein bands can be recognized, particularly, for the cultivation with LB medium, where rTfH and other protein bands appear simultaneously. In cultivations with the defined medium, the pattern looks differently. In the first phase, up to almost 4 h after induction, a single protein of about 57.4 kDa is found. By N-terminal sequencing, this band could be identified as the periplasmic located dipeptide binding protein (DBP), which is involved in the transport of dipeptides [19]. In the cultivation with complex medium, leakage of the outer membrane increased significantly 6 h after induction when C-starvation began, growth was slow but CO2 generation was still high. With the defined medium protein release in the extracellular medium was drastically rising at about 12 h after induction when all glucose was consumed. The findings shown in Fig. 3 are in accordance with the results reported by Rinas and Hoffmann [20], who studied in detail the leakage of host-cell proteins of recombinant and non-recombinant E. coli. These authors concluded that irrespective of the cell line, proteins of periplasmic origin are predominantly released in the medium, specifically, under stress conditions like temperature increase, nutrient starvation and high cell densities.
Fig. 3SDS-PAGE under denaturating conditions: EFs of different cultivations are shown after increasing the temperature to 39°C to induce rTfH production, a batch cultivation with complex medium, b batch cultivation with the defined medium, dashed line marks the beginning of glucose starvation
The SDS-PAGE shown in Fig. 3 indicates that the further protein bands observed for each media differ only in their intensity but not in their composition. This suggests that protein release in the medium cannot be attributed to cell lysis but is mainly due to an increasing leakage of proteins located and transported to the periplasm from the cytoplasm by the Sec-transport system.
This conclusion is also confirmed by analysis of the N-terminal sequence of rTfH, which clearly revealed that the OmpA leader sequence has been cleaved. However, cleavage was not uniform. Only 15% of the secreted rTfH found in the extracellular medium was cleaved at the expected cleavage site (Fig. 4). The remaining rTfH still carried parts of the OmpA leader peptide. The incomplete processing of signal sequences has been reported before [21]. The cleavage motif of a signal petidase is the A-X-A box [21] and fission occurs after the X. The N-terminal analysis of the secreted rTfH revealed that the cleavage motif is kept. These observations may allow the conclusion that the Sec-transportation pathway is overloaded, which might explain the improper cleavage of the signal peptidase.
Fig. 4Amino acid sequence of the signal peptide OmpA (underlined) and the beginning of rTfH; arrows mark the cleavage site and their relative frequency
Fed-batch cultivation
To enlarge volumetric rTfH activities, fed-batch cultivations were carried out. The basic assumption is that the volumetric productivity Qp [U (L h)−1] is given by equation  with the specific productivity qp [U (gDCW h)−1], which is anticipated to be approximately constant when increasing the biomass concentration. As observed in the controlled batch cultivation it was also thought that the target enzyme may be released into the cultivations medium from the periplasmic space under high cell density conditions, thus facilitating down-stream processing [20]. High cell density cultivation (HCDC) for E. coli can be effectively performed by using an appropriate defined medium. After the batch phase with unlimited growth and consumption of glucose, the cultivation is continued by feeding glucose at a feed rate resulting in a reduced growth rate (μset < μmax; Eq. 1). Thus, oxygen starvation can be prevented and the culture grows under glucose limitation in the feed phase. The oxygen partial pressure was kept constant at 20% as described in the Cultivation section. Two typical HCDC runs are shown in Fig. 5.
Fig. 5Expression of rTfH in E. coli TG1 during HCDC. Cells grew at 30°C before induction by shifting the temperature to 39°C (dashed line). a After 12.8 h, the batch phase ended and exponential feed was started and set to a growth rate 0.12 h−1. At the time point of 25.5 h, feeding was set to a constant rate (12.8 gglc h−1). b Fed batch with an exponential feeding profile with μset=0.12 h−1 over the entire feed phase including induction
Starting with an initial glucose concentrations of 24 g L−1, the biomass concentration is about 12 gDCW L−1 at the end of the batch phase (Fig. 5a, b). Due to the metabolic overflow, acetate was generated in the unlimited growth phase (μmax = 0.47 h−1), which was remetabolized at the end of the batch phase (Fig. 6). Then the glucose feed was started. The feed rate was adjusted to give a growth rate of about 0.12 h−1 according to Eq. 1, by assuming a yield coefficient YX/S of 0.4 g g−1. The actually measured growth rates were 0.09 (Fig. 5a, b). In Run A, after a feed phase of 12.8 h, a biomass concentration of 38.5 gDCW L−1 was obtained and at this point, the expression of rTfH was induced by shifting the temperature from 30 to 39°C within 15 min. The glucose feed rate was not further increased (12.8 gglc h−1) but kept constant to the value when the induction was started. Therefore, the growth rate steadily decreased and after about 14 h, the induction growth ceased. In the first phase after induction (14 h), an average growth rate of roughly 0.03 h−1 can be estimated from the data. However, over the entire period of 22.4 h with the constant feed rate, CO2-generation rate was also constant. Therefore, the fed glucose is used only for maintenance in the second part of the induction phase when there was zero growth . In the fed-batch phase of Run B, the same exponentially increasing glucose-feed rate as for Run A was applied, i.e., Fglc was calculated from Eq. 1 for μset = 0.12 h−1 and YX/S = 0.4 g g−1. After starting the induction at 42 g L−1, DCW exponential glucose feeding was continued for the same μset (0.12 h−1). In spite of the exponential glucose feeding, growth decreased and ceased about 6 h after induction. An average growth of 0.07 h−1 can be estimated from the experimental biomass data at the first 6 h of the induction phase. CO2 generation increased exponentially even when the growth was negligible in the second half of the induction phase. After 11.4 h induction, a final biomass concentration of 67.8 gDCW L−1 was obtained.
Fig. 6Glucose and metabolites formed during HCDC (Run A). cf. Fig. 5
In the course of both runs, the respiratory quotient was always about 1 (± 0.02). This indicates that consumed glucose was exclusively used for ATP production. Indeed, an intermediate formation of fermentative by-products such as acetate, succinate and formate was only observed at low level at the beginning and end of the induction period (Fig. 6). Both during the fed-batch and the induction phase, the glucose concentration was always zero. Carbon balances were checked by the installed computer program with consideration of volume changes by feeding and sampling and found to be close to  100 ± 2%.
The kinetics of the appearance of the heterologous gene product (rTfH) in the periplasm and cultivation broth is shown in Fig. 7 for Run A with a long induction phase (> 20 h). The activity in the periplasm steadily increased up to a value above 70 U mL−1 while in the first 10 h, no rTfH was released in the supernatant. The final activity in the medium was rather low (12 U mL−1). A similar release kinetics as shown in Fig. 7 was also observed for Run B, which differs largely from the behaviour found for batch cultures (Figs. 1, 2). In contrast to the batch cultivations the periplasmic activity does not pass a maximum and, particularly, little product (say less than 15%) is found in the medium.
Fig. 7Production kinetic of rTfH in the fed-batch cultivation (Run A). At a biomass of 38.5 gDCW L−1 rTfH expression was induced by raising the temperature to 39°C
However, despite the low leakage of rTfH into the medium a SDS-PAGE (Fig. 8) made from the supernatant after the enzyme induction showed that the cells were already leaky at the beginning of the induction, which is proven by the appearance of the periplasmic DBP. During the induction phase, an increase both of DBP and rTFH is observed.
Fig. 8SDS-PAGE: EFs of different cultivations are shown after increasing the temperature to 39°C to induce rTfH production, HCDC (Run A)
The hydrolase activity (pNPP test system) was also measured in the cytoplasm before the induction and at the end of the HCDC. While no activity was found prior to induction, a surprisingly high intracellular activity was detected at the end of the cultivation process. Indeed, about two-thirds of the entire rTfH activity was located in the cytoplasm, apparently in soluble and active form. Any significant inclusion-body formation was not observed. The operational parameters and the results received in the two HCDC runs shown in Fig. 5 are summarized in Table 2. In spite of the different feed rates and the total induction times, the rTfH activities found in the different compartments are in reasonable agreement. The constant feed rate after induction in Run A yields higher specific activities obviously due to lower biomass formation. However, as a result of the long induction time, the productivity is lower for Run A than for Run B. In comparison to the batch runs (Table 1) the accumulation of product in the periplasm and cytoplasm is most noticeable. Thus the total volumetric activity is about 30-fold larger than that achieved in the batch runs. These higher overall product concentrations are advantageously for rTfH recovery and purification.
Table 2Applied operation parameters and achieved rTfH activities in fed-batch runs (starting glucose concentration 24 g L−1, pH 7, T = 30°C, Tind = 39°C)Cultivation Run ARun BDCW at induction (g L−1)38.542Feed rateConstant (12.8 gglc h−1)Exponential (μ = 0.12 h−1)Estimated mean growth rate after induction (h−1)0.030.07Total induction time (h)22.411.4Final DCW (g L−1)53.267.8SupernatantVolumetric activity (103 U L−1)12.045.16Specific activity (103 U gDCW−1)0.2270.076PeriplasmVolumetric activity (103 U L−1)74.2251.69Specific activity (103 U gDCW−1)1.3950.762Cytoplasm (soluble)Volumetric activity (103 U L−1)141.24156.74Specific activity (103 U gDCW−1)2.6552.312TotalVolumetric activity (103 U L−1)227.5213.59Specific activity (103 U gDCW−1)4.283.15Productivity [U (103 gDCW h)−1]a0.190.28aTotal induction time
Purification
Two different strategies were applied to purify rTfH with regard to different uses. To gain rTfH with a high purification grade, the His6-affinity tag cloned at the C-terminal end of the bta1 gene allowed a simple and effective chromatographic procedure. rTfH was purified from the culture broth or the periplasm, which was obtained by an osmotic shock treatment (Localization of rTfH section). In the following, the purification of rTfH is described, which originated from the periplasmic space. About 37 g of wet biomass (corresponding to ca. 6.5 gDCW) was subjected to osmotic shock treatment (1 L scale). After separating the cells by centrifugation, 25 mL Ni2+ activated sepharose was directly added to the supernatant harbouring the periplasmic protein. After 1 h incubation, only low amounts of rTfH activity (0.1%) could be detected in the supernatant. The suspension was then filled into a glass column. Particle settling could be improved by connecting a peristaltic pump to the column outlet. After connecting the column to a FPLC system, rTfH was washed with bindung buffer; rTfH was eluted by applying a linear gradient of imidazole (Fig. 9a). rTfH elution was observed at an imidazole concentration of about 200 mM. In the pooled fractions, a rTfH activity of 6.53 U mL−1 could be detected. The His6-affinity step resulted in a 29-fold purification and a loss of about 30% rTfH activity.
Fig. 9Purification of rTfH from the PF. E. coli TG1 cells derived from a fed-batch cultivation (Run A); 37 g biomass was used for osmotic shock treatment, a Ni2+-affinity chromatography with Chelating Sepharose Fast Flow, b gelfiltration with Superdex 75 prep grade; pooled fraction are marked by a line
To further eliminate impurities and to remove high concentrations of NaCl and imidazole from the protein sample, gelfiltration was done using a Hi Load 26/60 Superdex 75 prep grade column with a 0.9% NaCl buffer (Fig. 9b). Before applying the sample to the column, pooled fractions with rTfH from the Ni2+-affinity chromatography were concentrated by ultrafiltration (concentration factor 7; cut-off 10 kDa). Gelfiltration assured purification to homogeneity as shown by SDS-PAGE (Fig. 10) although only a minor increase of the specific activity was observed (Table 3). Gel filtration resulted in a further loss of rTfH activity of about 10%. Totally, rTfH could be purified 30-fold with a 57% recovery in activity by the applied purification route. Table 3 gives an overview of the procedure to obtain the highly purified rTfH, which will be used for mechanistic studies (adsorption, kinetic) and structural analysis.
Fig. 10SDS-PAGE: rTfH at different purification steps (silver staining), 37 g biomass was used for the osmotic shock treatment, cells derived from HCDC (Run A) standard: molecular mass protein standard, 1 supernatant from the periplasm, 2 Ni2+-affinity chromatography, 3 pooled fraction after Superdex 75 gel chromatographyTable 3Summary of two routes used to recover and purify rTfH from HCDC; the highly purified product was obtained from the periplasmic fraction (Run A), the low grade product was recovered from the cytoplasm and periplasm by ultrasonification (Run B)Purification stepTotal units (103 U L−1)Total protein (mg L−1)Specific activity (U mg−1)Purification-factorYield (%)High grade rTfH Supernatant with periplasmic protein8.57587151100 Ni2+-affinity chromatography6.53154362971 Gel filtration4.89114453057Low grade rTfH Crude extract26.522,57010.321100 Heat treatment22.511,47015.31.585 Ultrasonification + heat treatment28.891,41020.52109aaCrude extract (ultrasonification plus centrifugation)
Contrary to the purification profile of the TfH from the culture broth of T. fusca cultivations, the overall yield could be increased from 14 to 57% [7]. Prior to performing the first purification step, the supernatant containing TfH had to be concentrated by ammonium sulphate precipitation. By this method, a final recovery of only 37% could be achieved because of the limited amounts of the precipitated proteins that could be redissolved [7]. By inserting the His6-affinity tag at the C-terminal end of rTfH, precipitation could be avoided and the recombinant enzyme could be easily obtained from E. coli-culture broths independently of its composition.
For industrial processes, purification with Ni2+-affinity chromatography is too costly of course. Therefore, an alternative purification route was developed, which aimed at a rTfH preparation of a low purification grade but sufficient activity and stability for larger-scale investigations on polyester degradation. Activity test of the soluble protein in the cytoplasm obtained during HCDC showed that most of the expressed rTfH was not secreted into the periplasm but remained in the cytoplasm. Hence, the cells were disrupted by ultrasonification to get rTfH both from the cytoplasm and the periplasm. In preliminary experiments, different ultrasonication conditions were tested. Three-fold pulses of 30 s each with 2 min break were found as the best treatment for the test conditions of 75 mg biomass resuspended in 2 mL PBS buffer. Here, biomass from the HCDC Run B was used. After the separation of cell debris by centrifugation, an activity of 26.52 U mL−1 was detected corresponding to a specific activity of 4.1 U gDCW−1. Chemical disruption of the cells by a detergent formulation (BugBuster, see Localization of rTfH section) resulted only in a specific activity of 3.07 U gDCW−1 implying that cell disruption is more effective than the chemical treatment. Protein concentration of the crude extract was 2.57 mg mL−1. Taking the specific activity of the high-grade rTfH into account (445 U mgProtein−1), 1 mg protein harboured 2.3% heterologous produced rTfH. Because rTfH is very stable against higher temperatures [7], heat treatment was carried out at 55°C for 10 min. Two routes were tested, namely either heat treatment of the disrupted cells immediately after the ultrasonication or heat treatment after the separation of the cell debris. Activity tests revealed that a heat treatment directly after the cell disruption yielded the best results. Thus the purification factor could be increased from 1.5 to 2 and a higher yield was observed. Table 3 summarized the results in detail.
Discussion
The recombinant strain E. coli TG1 harbouring the plasmid pCYTEXP1-OmpA-bta1-His6 proved to be an appropriate system for expression, secretion and purification of the hydrolase from T. fusca. In comparison to cultivations with T. fusca and induction of TfH by adding polyesters a 1.5–2-fold higher volumetric activity could be obtained in batch cultivations of the recombinant E. coli strain. The volumetric activity could be further improved in fed-batch cultivations with glucose limitation. Considering only the extracellular and PFs, the increase is 20-fold. If one also accounts for the activity found in the cytoplasm, the total volumetric activity is over 50-fold higher than that obtained from T. fusca. The overall volumetric productivity [U (L h)−1] increased by a factor more than 100. Hence, the production of rTfH in recombinant E. coli is an attractive and promising approach for providing hydrolytic activity for polyester cleavage in degradation and recycling processes. This view is supported by the simple recovery route to give a crude and stable rTfH preparation, which did not exhibit activity losses after storage at −20°C for several weeks.
A further improvement of rTfH production in E. coli is the insertion of the His6 tag at the C-terminal end of the enzyme. Purification of the wild-type TfH proved to be quite ineffective. A three-step purification strategy (precipitation, ion-exchange chromatography and hydrophobic interaction chromatography) resulted in only 14% yield in activity [7]. With the His6 tag, 57% activity yield of rTfH could be obtained in a two-step purification route starting from the supernatant and PFs. A 30-fold purification was achieved yielding a specific activity of 445 U mg−1. From this value, the overall concentration of the produced rTfH can be estimated as about 0.5 g L−1.
In the batch runs with LB and the defined medium, better results were found by induction to only 39°C. Therefore, fed-batch cultivations were carried out with this induction temperature. While both the media yielded comparable volumetric activities, the specific activities differ significantly (about fivefold in LB). LB medium obviously supplies E. coli with all nutrients to easily synthesize the recombinant protein at high specific productivity and to secrete it across the cell envelope. Increased growth rates and facilitated protein production in LB medium are frequently reported in the literature [22, 23]. In batch cultivations with both media, rTfH expression as well as its transport across the cell membrane in the periplasm and the subsequent release into the cultivation media are seemingly coupled with growth and CO2 generation (Figs. 1, 2).
In the fed-batch cultivations with the defined medium, roughly tenfold higher activities could be achieved in the supernatant and periplasm than in batch operation. Surprisingly, the specific activities in the glucose limited fed-batch process are even twofold higher. If one accounts for the soluble cytosolic fraction the activity yields further improve remarkably. The total specific activity of the HCDCs is in the same range as those obtained for the batch process with LB medium. An influence of the His6 tag and the full or truncated OmpA leader sequence on TfH activity can hardly be estimated from the results of this study. However, the high activities of rTfH obtained suggest that small sequences at the C- and N- terminals might have little effect on TfH activity. The fraction of soluble active protein located in the cytoplasm is remarkably high but its amount can be estimated to be less than 2% of the total protein content of the cells (assuming 50% protein of DCW). Therefore, it is understood that inclusion body formation could not be detected.
Among the different mechanisms to transport protein from the cytoplasm across the cell membrane to the periplasm, the Sec pathway is the most prominent for prokaryotic organisms [24]. The Sec-translocation process is thought to be generally responsible for the transport of newly synthesized but unfolded proteins out of the cytosol to the periplasm. This process is accompanied by the cleavage of the leader sequence (OmpA) from the pre-protein and proper folding in the periplasm. If we assume that the Sec pathway is also active for the transport of the heterologous rTfH out of the cytoplasm, it is obvious that due to its accumulation in the cytoplasm during the fed-batch runs, the translocation rate is less than the expression rate. Hence, transport into the periplasm apparently presents a limiting step. It might be possible that the entire translocation process is subjected to an equilibrium phenomenon in as far as high protein concentrations in the periplasm reduces the transport rate. This is evident if the rTfH masses (or units) in periplasm and cytoplasm are not referred to cultivation volume as given in Table 2 but to their respective actual volumes. If it is assumed that the volume of periplasm is about 5% of the cell volume then the rTfH concentration in the periplasm is more than tenfold higher than in the cytoplasm. This might explain that no cytoplasmic rTfH activity was found in the batch runs, where the periplasmic protein was entirely released in the extracellular space. In contrast, relatively little protein was transported across the outer cell wall into the medium in the fed-batch runs with high cell densities and higher rTfH activities were found in both the cytoplasm and periplasm.
As to the Sec pathway, the rTfH in the cytoplasm should be unfolded and hence is not expected to be in the active form. However, when gaining the cytoplasmic fraction (see Localization of rTfH section) immediate activity was observed. This suggests that either folding takes place spontaneously in the PBS buffer or the cytosol already harbours the active folded rTfH and its translocation follows another mechanism. Unfortunately, our study does not allow to distinguish between the two possibilities.
Though it is often assumed that E. coli laboratory strains do not secrete proteins across the cell envelope in the extracellular medium at normal growth conditions [25], leakage of the outer cell membrane is a common phenomenon observed for a variety of cultivation conditions as reported in recent papers [20, 25, 26] and review articles [21, 27]. Permeability of the outer cell wall may depend on medium composition, growth rates, cell concentration, changes in cultivation conditions, etc. For instance, Mg2+ and Ca2+ increase leakage of the outer membrane [28]. Rise of temperature causes changes in membrane permeability and fluidity due to alterations in the lipid composition of the cell wall. This aspect was particularly studied in continuous cultures of E. coli [26] and an increased permeability was found at higher growth rates. Rinas and Hoffmann [20] studied in detail the leakage of host-cell proteins during HCDCs of recombinant and non-recombinant E. coli. These authors reported that the proteins found extracellularly are of periplasmic origin except the heat-shock protein DnaK. Various and interrelated stress factors in the temperature-induced HCDC are suggested to be responsible for the migration of periplasmic proteins in the extracellular medium.
In all cultivations performed in this study, rTfH could be detected in the medium without applying any additional measures to permeabilize the outer cell membrane. Cell lysis as a possible mechanism of rTfH release can be excluded as the cleavage of the OmpA sequence in the extracellularly found hydrolase indicates transport across the cytoplasmic membrane by the Sec-translocation mechanism. Any significant cell lysis would also release cytoplasmic rTfH still containing the complete signal sequence.
In batch cultivations (Figs. 1, 2) with LB and the defined medium, induction of rTfH expression was started at low cell densities. Immediately after the temperature upshift, rTfH appeared in the periplasm but its periplasmic activity passes a maximal value just in the first part of the exponential growth phase. In accordance with the findings of Shokri et al. [26], the migration of the periplasmic rTfH in the medium is obviously growth coupled. This effect may be attributed to changes in cell-wall composition which facilitate transport into the extracellular medium. On the other hand, during cell growth and cell-wall biosynthesis, transient openings of the outer membrane and the murein layer are unavoidable [29], thus the protein of periplasmic origin can migrate into the extracellular medium. Furthermore, vesiculation and fragmentation of the outer membrane may occur as a natural phenomenon during the exponential growth phase [30, 31] which is thought to be caused by an imbalance of the outer-membrane biogenesis and peptidoglucan synthesis. Therefore, several growth-coupled phenomena may contribute to the migration of periplasmic proteins in the medium.
At the end of the batch cultivations, the produced rTfH has entirely accumulated in the medium. As can be discerned from the SDS-PAGE shown in Fig. 3 the transport of rTfH across the cell envelope is accompanied simultaneously by release of other periplasmic proteins [20]. The kinetics of rTfH accumulation in the extracellular medium (Figs. 1, 2) agrees qualitatively with the results shown in Fig. 3, particularly, no significant migration is observed at low growth rates after the induction when the culture adapts to the higher temperature. Rinas and Hoffmann [20] found DBP as the most prominent periplasmic protein transported to the medium. This observation and even the release kinetics reported by these authors for high cell-density conditions are in striking agreement with our findings for batch conditions (relatively low cell densities) as observed in the defined medium (Fig. 3b).
In contrast to the batch runs, rTfH accumulates in the cytoplasm and periplasm during the glucose-limited fed-batch cultivations after induction at higher cell densities. The relative fractions of rTfH released extracellularly is low. For an induction period of 11 h, only 5 and 10% of the PF are released for Run A and B, respectively. As in Run B, the growth rate was higher as a result of exponential glucose feeding; this finding underpins the view that the permeability of the outer membrane is related to growth rate. As shown in Fig. 8 the outer membrane is already leaky before the temperature shift from 30 to 39°C as among other periplasmic proteins the DBP is predominantly found in the extracellular medium as reported previously [20]. Of course, in the induction phase a further increase of DBP is observed (Fig. 8). Though the transport of rTfH in the periplasm as well as its migration into the extracellular medium continued (Fig. 7), the cultivation was terminated to prevent cell lysis after growth ceased. However, in the non-growth phase, glucose does not accumulate in the medium but is consumed as is also exhibited by the monotonous increase of CO2 generation. This points to the increased maintenance and ATP demand for translocation of the heterologous gene product (rTfH) across the inner cell membrane.
These mechanisms, which govern the local distribution of rTfH are not yet understood. Though cell-specific productivities are in a comparable range, product distributions differ largely. Our results suggest that the outer membrane permeability is related with the growth rate and hence all rTfH can migrate into the extracellular medium at high growth rates (batch runs). Therefore, final product release across the cell envelope could play an important role in the overall translocation process as product accumulation in the periplasm seemingly reduces the translocation rate from the cytoplasm by the Sec pathway. This hypothesis requires further investigations, which are presently in preparation. Therefrom, new strategies for cultivation and downstream processing with improved product qualities are expected.

purification
batch culture
sec pathway
recombinant protein expression
fed-batch culture


Effects of dopaminergic modulation on electrophysiological brain response to affective stimuli

Introduction Several theoretical accounts of the role of dopamine suggest that dopamine has an influence on the processing of affective stimuli. There is some indirect evidence for this from studies showing an association between the treatment with dopaminergic agents and self-reported affect.
Introduction
Dopaminergic neurotransmission has been suggested to be involved in several functions of the brain that are associated with reward, such as the experience of rewarding feelings (Wise and Bozarth 1985), reward-related reversal learning (Mitul et al. 2001), motivational aspects of reward (Robinson and Berridge 1993), and reward processing (Day and Carelli 2007). In addition, dopamine neurons mediate reward signals in appetitive learning of visual stimuli (Schultz 1997). Recently, Robbins and Everitt (2007) stated that “the role of enhanced DA activity is to increase responsivity to cues paired with reinforcement”. In addition to these functions, there are suggestions that dopamine is involved in the regulation of affect (Saeedi et al. 2006). Most studies addressing the role of dopamine in affect-regulation have been carried out among animals. Few studies address the role of dopamine in affect among humans. Studies among Parkinson’s disease patients, whose mesencephalic dopamine activity is decreased, show that dopamine is able to modulate emotional responding (Wieser et al. 2006). In addition, it has been demonstrated that dopamine is also involved in the perceptual processing of fearful stimuli in Parkinson disease patients (Tessitore et al. 2002). No studies are known which address whether dopamine is able to modulate the processing of affective stimuli in normal subjects. However, there are some indirect suggestions pointing towards a role of dopamine in affective processing. Takahashi et al. (2005) showed among healthy volunteers watching unpleasant pictures that the dopamine D2 receptor antagonist sultopride decreased responses in the limbic areas (amygdala, hippocampus, anterior cingulate), which are associated with affective processing. In contrast, they found that sultopride produced greater activations in the cortical areas (frontal, temporal, and parietal), and no effect of sultopride on self-reported affect was found, suggesting a complex role of dopamine in affective processing.
With regard to psychopathology, what several theoretical accounts of psychopathological behavior have in common is that they state that dopamine may have a function in signaling relevant or salient information (Franken 2003; Kapur 2003; Robinson and Berridge 1993) and suggest that dopamine is involved in the processing of information having motivational relevance. In concordance with this notion, Franken et al. (2004) showed that the attention-capturing properties of heroin-related stimuli in heroin-dependent patients were decreased as result of treatment with the dopamine antagonist haloperidol. Indirect evidence that dopamine is involved in the processing of affective information comes from a study of Taylor et al. (2005) among schizophrenics. They found that structures with extensive dopaminergic innervations, such as the ventral striatum, show blunted responses to emotional salience in schizophrenics.
In addition, several theories suggest that dopamine is involved in the experience of self-reported positive affect (Ashby et al. 1999; Burgdorf and Panksepp 2006). In concordance with these notions, several studies examining the effects of dopamine-antagonists, in particular D2 antagonists such as haloperidol, report a blunting of the self-reported affective response (e.g., Mizrahi et al. 2007). In contrast, dopamine agonists result in self-reported enhanced mood (Saeedi et al. 2006; Willner et al. 2005).
In the present study, we use event-related potentials (ERPs) to investigate the processing of affective stimuli, in particular reward-associated stimuli, in humans. ERPs measured during the perception of pictures are known to vary with their affective contents (Cacioppo et al. 1994; Cuthbert et al. 2000; Keil et al. 2001; Schupp et al. 2000). Affective pictures, both pleasant and unpleasant, elicit larger late positive potentials (LPP; starting 300–400 ms after picture onset until picture offset) than neutral pictures. LPPs capture controlled processes and are generated by a diffuse network including anterior areas such as the orbitofrontal cortex (Rule et al. 2002; Vuilleumier 2005) and higher order visual areas such as parietal and occipito-temporal regions (Keil et al. 2002). In addition to these late effects, the viewing of affective pictures results in reduced ERP amplitudes in the early time window (150–300) as compared to neutral stimuli (Codispoti et al. 2007; Schupp et al. 2006). This early ERP effect is sometimes referred to as the early posterior negativity (EPN; Schupp et al. 2003b). The EPN, which is associated with the perceptual encoding of emotional material, may reflect the call for resources in the capacity-limited second stage of processing (Codispoti et al. 2007; Schupp et al. 2006) and is generated in the visual occipito-temporal regions (Schupp et al. 2006).
In passive viewing contexts such as in the present study, the EPN and LPP modulation by affective stimuli are similar in respect to the fact that they are both pronounced for high arousing pleasant and unpleasant stimuli (Schupp et al. 2006). However, it seems that the LPP, in contrast to the EPN, is enlarged when allocating top-down attentional resources to emotion, suggesting that the EPN is relatively insensitive to voluntarily attentional processes. Previous studies show that similar designs that study the electrophysiological activation to affective pictures are able to measure the modulation of affective processing by psychopharmacological agents (Franken et al. 2007; Kemp et al. 2004).
This study will investigate whether a D2 agonist or a D2 antagonist can modulate the electrophysiological response to affective pictures. Decrease in dopaminergic activity will be induced by a single oral dose of 2 mg haloperidol. This dose does not produce serious side effects (Franken et al. 2004; Kahkonen et al. 2001), and 2 mg was shown to be able to induce modulation of the processing of neutral and rewarding information (Franken et al. 2004; Kahkonen et al. 2001). Increase in dopamine will be induced by a single oral dose of 2.5 mg bromocriptine. Generally spoken, there are several indications that bromocriptine can modulate the response towards rewarding stimuli (Kirsch et al. 2006).
As dopamine is associated with an enhanced signalling of reward-related stimuli, it was expected that bromocriptine enhanced the processing of pleasant stimuli and haloperidol decreased the processing of pleasant stimuli. Based on the literature, the hypotheses for unpleasant stimuli could be less clearly formulated. Because some studies show that dopamine only signals stimuli associated with reward while others do find that dopamine also signals punishment-related stimuli, we expected that bromocriptine decreased the processing of unpleasant stimuli and haloperidol enhanced the processing of unpleasant stimuli. Neutral stimuli served as control conditions.
Materials and methods
Subjects and procedure
A randomized, double-blind, placebo-controlled crossover design was employed. Participants were 32 healthy right-handed male undergraduate psychology students and were recruited by advertisement at the university campus. All participants were non-smokers. Eleven participants could not be included in the analysis because of excessive electroencephalographic (EEG) artifacts in one of the three sessions (n = 3), drop out because of side effects (nausea) in the bromocriptine treatment (n = 6), or drop out because of side effects (low blood pressure, n = 1 and nausea, n = 1) in the placebo treatment. In total, 21 participants completed all three the measurement sessions, and all analyses were conducted using this sample. The mean age of the subjects was 19.8 years (SD = 1.2, range = 18–22).
Two weeks before the experiments, participants who were willing to participate were informed about the procedure. One week before the start of the experiments, subjects underwent a clinical psychiatric and physical examination by an experienced psychiatrist (L.P.). Eligible participants were instructed to take the medication at 8:00 a.m., 10:00 a.m., or 12:00 a.m. 4 h before each session in the laboratory. Further, they were instructed to abstain from coffee, grapefruit, alcohol, and drugs at the days of the experiment. In addition, they were instructed to abstain from alcohol and drugs the day before the experiment. The time between each session was always 1 week. All participants received a financial compensation of 50 euro. This study was approved by the Medical Ethics Committee of the Erasmus Medical Centre, Rotterdam.
Treatment
All subjects received a single oral dose of placebo (lactose), bromocriptine (2.5 mg), and haloperidol (2 mg) in a counterbalanced order. The medication was provided by the pharmacy of the Erasmus Medical Centre in indistinguishable capsules. It has been demonstrated that a 2-mg dose of haloperidol does not produce serious side effects in healthy subjects (Franken et al. 2004; Kahkonen et al. 2001). Further, 2.5 mg of bromocriptine has been shown to modulate cognitive processes and in absence of side effects. Based on the literature, it was not expected that the 2.5 mg of bromocriptine would result in serious side effects (Jarvik et al. 2000). However, after several sessions, we noticed nausea occurring in some sessions. Because we excluded all participants with nausea (with reference to the possible interaction between nausea and the viewing of affective pictures), this resulted in relatively high dropout rates. After inclusion of 15 participants, all participants in all sessions were provided with domperidone (10 mg) to prevent nausea. Participants were instructed to take the domperidone together with the research medication1.
Stimuli and task
Before the first session, participants were instructed, and they signed informed consent. Subsequently, participants filled out the Positive and Negative Affect Scales (PANAS) questionnaire and two single-item self-rating scales that were used to assess the subjective effects of the medication (see “Questionnaires” section). After this, the participants were seated on a comfortable chair in a light and sound-attenuated room. Stimuli were presented on a 21″-monitor 1.5 m away from the eyes. After attaching the electrodes (approximately 15 min), they were instructed to pay close attention to the pictures that would be presented. To be sure that they paid attention to the pictures, they were told that questions about the pictures would be asked after the experiment.
All pictures were taken from the International Affective Picture System (IAPS; Lang et al. 1999). To reduce test–retest recognition effects, three pictures sets which were closely matched on valence and arousal ratings were used in a counterbalanced order (see Table 1). A one-way analysis of variance (ANOVA) confirmed that there were no differences in arousal and valence ratings between the three sets. Further, a repeated measures ANOVA confirmed that there were significant valence rating differences between the three affective categories, F(2,118) = 1324.2; p < 0.001. As expected, valence ratings for pleasant stimuli were higher than for neutral stimuli, and for neutral stimuli higher than unpleasant stimuli (higher scores indexes more pleasantness). In addition, analysis of the arousal ratings showed also significant differences between the three affective categories, F(2,118) = 304.5; p < 0.001. Both pleasant and unpleasant pictures had higher arousal ratings than neutral pictures. In addition, pleasant stimuli had slightly higher arousal ratings as compared to the unpleasant stimuli. The neutral pictures consisted mainly of buildings, household objects, and neutral persons. The pleasant pictures consisted mainly of erotic and romantic scenes, while the unpleasant pictures consisted mainly of mutilated bodies and threat scenes.
Table 1Mean (SD) valence and arousal ratings of the three sets of IAPS pictures according to the normative ratings of the IAPS database (Lang et al. 1999) Set 1Set 2Set 3Differences between sets (F values, ns)Neutral valence4.9 (0.3)4.9 (0.4)4.9 (0.2)0.01Arousal2.7 (0.4)2.8 (0.5)2.7 (0.6)0.16Pleasant valence7.2 (0.6)7.2 (0.6)7.2 (0.6)0.06Arousal6.3 (0.9)6.5 (0.7)6.2 (1.1)0.58Unpleasant valence2.5 (0.6)2.6 (0.5)2.6 (0.7)0.05Arousal5.8 (1.0)5.6 (0.9)5.7 (0.9)0.11ns Not significant
Questionnaires
The PANAS (Watson et al. 1988) were administered as a measure of positive and negative affect. The PANAS is a 20-item bidimensional mood inventory. Positive affect reflects the extent to which a person feels enthusiastic, active, and alert (Watson et al. 1988). Negative affect is a general dimension of subjective distress and unpleasurable engagement that subsumes a variety of aversive mood states including anger, contempt, disgust, guilt, fear, and nervousness (Watson et al. 1988). Psychometric properties of the PANAS scales are good (Boon and Peeters 1999; Watson et al. 1988).
Two single-item self-rating scales were used to assess the subjective effects of the medication. A five-point Likert scale (1 = no effect, 5 = a very strong effect) was used to assess the magnitude of the effect. A visual analogue scale (0 = very unpleasant, 100 = very pleasant) was used to assess the pleasantness of the effect.
Electroencephalographic recording
The electrophysiological signals were recorded through the Active-Two amplifier system (Biosemi, Amsterdam, The Netherlands) from 64 scalp electrodes (10–20 system) using Ag/AgCl electrodes mounted in an elastic cap. Furthermore, six additional electrodes were attached to left and right mastoids, two outer canthi of both eyes (HEOG), infraorbital, and supraorbital regions of the eye (VEOG). Two additional scalp electrodes were used as reference and ground electrodes, respectively. Online signals were recorded from DC to 134 Hz. All signals were digitized with a sample rate of 512 Hz and 24-bit A/D conversion. Data were off-line re-referenced to an average reference. Off-line, EEG and EOG activity was filtered with a bandpass of 0.05–30 Hz (phase shift-free Butterworth filters; 24 dB/octave slope). After ocular correction (Gratton et al. 1983), epochs including an EEG signal exceeding ±75 μV were not included in the average. The mean 200-ms pre-stimulus period served as baseline. After baseline correction, average ERP waves were calculated for artifact-free trials at each scalp site across the three stimulus conditions and across the three treatment conditions. Brainvision Analyzer (Brain Products, München, Germany) software was used for all EEG analyses.
Data analysis
Definitions of the peaks of interest were based on previous studies using the same IAPS paradigm (Franken et al. 2007; Schupp et al. 2000, 2003a). Inspection of voltage maps of the difference waves (emotional–neutral) revealed that the EPN was maximal present at the following posterior sites: P5/6, P7/8, P9/10, PO3/4, PO7/8, and O1/2 (see Fig. 1). This site is comparable to that used by previous studies (Franken et al. 2007; Schupp et al. 2003b). To reduce the number of covarying variables and to reduce the possible influence of single spurious electrodes, these individual sites were collapsed by averaging the electrodes into one region per hemisphere (Dien and Santuzzi 2005; Schupp et al. 2003b).2 The EPN in this area was defined as the mean value of ERP activities in 150- to 300-ms time windows. Note that the EPN can have positive values: The EPN is a negative going potential showing reduced positivity for emotional stimuli.
Fig. 1Scalp topography of the EPN component (mean value in the 150- to 300-ms time window). Voltages represent difference scores: Pleasant–neutral (left panel) and unpleasant–neutral (right panel). Note that the anterior activity is positive and posterior activity is negative
Inspection of voltage maps of the difference waves (emotional–neutral) revealed that the LPP was maximal at central and centro-parietal sites C5/6, C3/4, C1/2, CP5/6, CP3/4, CP1/2, P5/6, P3/4, P1/2 (see Fig. 2). Again, these individual sites were collapsed by averaging these electrodes into one region per hemisphere. The LPP was defined as mean value of ERP response within 400–700 ms. The LPP is a positive deflection in the EEG, and emotional stimuli increase the amplitude of this component. In addition, because in previous studies valence effects on the LPP have shown hemispheric differences (e.g., Keil et al. 2001), we exploratively included hemisphere as factor in all ANOVAs.
Fig. 2Scalp topography of the LPP component (mean value in the 400- to 700-ms time window). Voltages represent difference scores: Pleasant–neutral (left panel) and unpleasant–neutral (right panel). Note that the anterior and posterior activities are negative and that the central activity is positive
Statistical analysis
To test the influence of dopaminergic manipulation on the EPN and LPP components, a 3 (medication: placebo, bromocriptine, haloperidol) × 3 (stimulus category: neutral, pleasant, unpleasant) × 2 (hemisphere: left, right) repeated measures ANOVA was employed with all variables as within-subjects factors. For the manipulation check, a three-way repeated measures ANOVA was used for each self-reported measure (pleasantness and magnitude of medication effect, positive and negative affect). In all instances, Greenhouse–Geisser adjusted p values were used. In case of a significant ANOVA result, partial Eta squared (η2) as measure of effect-size is reported. Significant ANOVA effects were followed up by pairwise comparisons with Bonferroni adjustment.
Results
Self-reported effects
There was no significant effect of treatment on the magnitude of the subjective effects of the medication, F(2,40) = 0.8; p = 0.45. In addition, no differences in experienced pleasantness of the medication between the conditions was observed F(2,40) = 0.0; p = 0.96. Further, no effects of the medication condition on negative, F(2,40) = 0.8; p = 0.45, nor positive, F(2,40) = 0.4; p = 0.66, affect was observed. Summarized, no self-reported effects of medication on magnitude and pleasantness of effects and affect were observed.
ERP data
EPN component
Table 2 displays all mean values of the EPN and LPP components, including standard deviations. Figs. 3, 4, 5, and 6 show the difference ERP waves (emotional minus neutral) for affective stimuli at the left and right hemisphere sites. As expected, a significant main effect for stimulus category was observed, F(2, 40) = 81.0, p < 0.001, η2 = 0.80. Pairwise follow-up analysis show that both pleasant (p < 0.001) and unpleasant stimuli (p < 0.001) yielded smaller ERP values as compared to neutral stimuli, suggesting an enhanced early perceptual encoding of affective stimuli. However, no significant medication × stimulus category interaction effect was observed F(4, 80) = 0.17, ns, suggesting that dopaminergic medication had no influence on this early stage of picture processing. In addition, no other significant main or interaction effects were observed.
Table 2Mean (SD) values of the EPN and LPP components in left and right hemisphere towards neutral, pleasant, and unpleasant pictures for the placebo, bromocriptine, and haloperidol treatment conditions (n = 21) PlaceboBromocriptineHaloperidolEPN neutral left9.4 (3.6)8.4 (3.7)9.4 (4.4)Right8.6 (3.1)8.5 (4.2)8.9 (3.6)EPN pleasant left7.0 (4.3)6.0 (4.0)6.9 (4.2)Right6.7 (3.8)6.3 (4.5)6.8 (3.8)EPN unpleasant left8.6 (4.0)8.0 (3.8)8.6 (4.4)Right8.5 (4.0)8.2 (4.4)8.6 (4.3)LPP neutral left3.5 (1.6)3.2 (1.9)3.4 (1.8)Right3.8 (1.4)3.1 (1.7)3.3 (1.7)LPP pleasant left4.9 (1.9)4.6 (1.8)4.8 (2.1)Right4.6 (2.0)4.2 (1.8)4.2 (1.7)LPP unpleasant left5.4 (1.7)4.7 (1.7)5.5 (2.0)Right4.8 (1.9)4.2 (1.8)4.5 (1.9)Fig. 3Differences waves (pleasant–neutral) for pleasant stimuli at the left and right posterior cluster (P5/6, P7/8, P9/10, PO3/4, PO7/8, and O1/2) for placebo (black line), bromocriptine (gray line), and haloperidol (dotted line) treatment conditions separatelyFig. 4Differences waves (unpleasant–neutral) for unpleasant stimuli at the left and right posterior cluster (P5/6, P7/8, P9/10, PO3/4, PO7/8, and O1/2) for placebo (black line), bromocriptine (gray line), and haloperidol (dotted line) treatment conditions separatelyFig. 5Differences waves (pleasant–neutral) for pleasant stimuli at the left and right central/centro-parietal clusters (C5/6, C3/4, C1/2, CP5/6, CP3/4, CP1/2, P5/6, P3/4, P1/2) for placebo (black line), bromocriptine (gray line), and haloperidol (dotted line) treatment conditions separatelyFig. 6Differences waves (unpleasant–neutral) for unpleasant stimuli at the left and right central clusters (C5/6, C3/4, C1/2, CP5/6, CP3/4, CP1/2, P5/6, P3/4, P1/2) for placebo (black line), bromocriptine (gray line), and haloperidol (dotted line) treatment conditions separately
LPP component
Again, a significant main effect for stimulus category was observed, F(2, 40) = 48.1, p < 0.001, η2 = 0.71. Pairwise follow-up analysis show that both pleasant (p < 0.001) and unpleasant stimuli (p < 0.001) yielded a larger LPP component as compared to neutral stimuli, suggesting an enhanced processing for affective stimuli in the late elaborative phase. In addition, a small but significant main effect of medication was found, F(2, 40) = 5.2, p < 0.05, η2 = 0.21. Follow-up analysis indicated that bromocriptine resulted in overall reduced LPP waves as compared to the placebo treatment (p < 0.05), suggesting that bromocriptine results in an overall reduced processing of pictorial stimuli in this stage. Further, a small but significant stimulus-category × hemisphere interaction effect was observed, F(2, 40) = 3.7, p < 0.05, η2 = 0.16. Because none of the post hoc tests resulted in a significant pairwise comparison, and this interaction had not our major interest, we will not discuss this finding in further detail. Most important for our hypothesis, no significant stimulus category × medication interaction effect was found, suggesting that the employed dopamine antagonist and agonist had no selective influence on affective processing.
Discussion
To our knowledge, this is the first study examining the influence of dopamine on ERP correlates of affective stimulus processing. The present study demonstrates that an acute low dose of bromocriptine resulted in globally reduced LPP waves as compared to the placebo treatment, suggesting that bromocriptine results in decreased cortical processing of pictorial information. Although there are no previous studies using an acute low dose of bromocriptine examining the processing of emotional pictures (as in the present study), these results are in line with a study of Oranje et al. (2004). Although in that study a different paradigm was employed and different components were studied, these authors found a globally reduced ERP response as result of bromocriptine treatment. In contrast to bromocriptine, an acute low dose haloperidol did not result in a non-specific modification of cortical processing of pictorial information. Previous studies addressing information processing using haloperidol and ERP measures yielded mixed results. For example, Kahkonen et al. (2001) did observe a global reduction of ERP waves using 2 mg (oral) of haloperidol. In contrast, other studies did not observe a general effect of haloperidol on ERP amplitudes (e.g. Ford et al. 1994). Clearly, the effects of haloperidol on ERPs are dependent on dose, ERP component, characteristics of the participants, and experimental paradigms that are used.
Most importantly, we could not confirm the hypothesis that D2 receptor modulation results in a modulation of cortical processing of affective stimuli. Neither an acute low dose of bromocriptine nor an acute low dose of haloperidol did differentially modulate the brain’s electrophysiological response towards affective stimuli. This finding suggests that dopamine D2 receptors are not involved in the cortical processing of affective information. Because the studied ERP components measured in the present study are associated with attentive motivational processing (Schupp et al. 2006, 2007), it can be concluded that motivational attentive processing is not affected by haloperidol nor bromocriptine. Given that the ERPs in the present study reflect attentive processing, the results are in line with Oranje et al. (2006) who did not find any effects of l-dopa nor bromocriptine on ERP indices of selective attention in healthy volunteers.
In addition to the main finding that modulation of the D2 receptor did not result in early (EPN) or late (LPP) effect on ERPs indexing early perceptual encoding of emotional material and subsequent elaborative processing, we did not observe an effect of haloperidol and bromocriptine on the subjective self-reported affect. Neither medication had an effect on positive or negative affect. However, it must be noted that the present study was not specifically designed to measure the subjective effects of the medication. In addition, although the present results show that dopamine is not associated with the perception of reward-associated stimuli per se, it still might be associated with establishing stimulus–reward associations or functions dealing with reward-related motor responses. In addition, it might be that the reward processing is only modulated by dopaminergic agents in persons with a genetically heightened reward sensitivity, for example, carriers of the A1 allele of the dopamine D2 TaqIA gene (Kirsch et al. 2006).
The present study suggests that low doses of bromocriptine in Parkinson’s disease patients and haloperidol in schizophrenia patients may have limited influence on affective processing and self-reported affect in patients having these disorders. However, it must be noted that we did only study the effects of an acute dose of these medications; it is not known whether these effects differ from those associated with long term use of medication.
The present study has several limitations. First, there was a substantial dropout in the bromocriptine treatment. It may be that this is a specific subgroup of persons with heightened sensitivity for dopamine agonists. Because this group is not included in the analysis, this might have resulted in lowered generalizability of the results. Second, because of risks of unwanted side effects, we employed rather low doses of bromocriptine and haloperidol, 2 mg and 2.5 mg, respectively. Third, a possible limitation of this study is that some participants received domperidone and others did not, which may have influenced the results. However, it must be noted that domperidone does not cross the blood–brain barrier but exerts its effect peripherally, and as such, does not have a major influence on the central dopamine system (Barone 1999; Champion et al. 1986). In addition, additional statistical analyses show that domperidone did not influence any of the outcome variables (electrophysiological nor self-reported) used in the present study.
Summarizing, the present results indicate that neither an acute low dose of bromocriptine nor an acute low dose of haloperidol has selective effect on electrophysiological indices of affective processing and self-reported affect. In contrast, bromocriptine decreased overall processing of all stimulus categories regardless of their affective content. The results indicate that dopaminergic D2 receptors do not seem to play a crucial role in the selective processing of affective visual stimuli.

dopamine
affective processing
haloperidol
erp
bromocriptine