3.2 Materials and methods
with 180 ml MEC buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) at 1 ml/min. The resin was subsequently calibrated with a calibration solution [5 mg each of blue dextran (200 kDa), sheep IgG (130 kDa), bovine serum albumin (BSA, 68 kDa), ovalbumin (45 kDa) and myoglobin (14 kDa) in MEC buffer (2 ml)]. The resin was eluted at 0.08 kPa with a flow rate of 0.5 ml/min, with 1 ml fractions collected and the absorbance being recorded at 280 nm. The resins were regenerated with 60 ml of a NaOH solution (0.2 M) and re-equilibrated with 180 ml MEC buffer. The Fischer plots were constructed for the protein calibration solution whereby the log of the respective molecular weights were related to their respective elution volumes for the S300 (Fig.
3.1) and the S200 (Fig. 3.2) HR molecular exclusion resins. The elution volume (Ve) or retention volume of blue dextran was taken as the void volume (V0) of the column.
Figure 3.1: Calibration curve of a HiPrep™ 16/16 Sephacryl™ S300 HR molecular exclusion resin. A HiPrep™ 16/16 Sephacryl™ S300 HR resin (16 x 600 mm, flow rate of 0.5 min/min, 0.08MPa) was calibrated with a calibration solution in MEC buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). Fractions were monitored by recording the absorbance at 280 nm and the resulting relative mobility of the component protein plotted against the log of the respective molecular weights. The equation of the trendline is given by y = - 40.03x + 134.2, with a correlation coefficient of 0.984.
Figure 3.2: Calibration curve of a HiPrep™ 16/16 Sephacryl™ S200 HR molecular exclusion resin. A HiPrep™ 16/16 Sephacryl™ S200 HR resin (16 x 600 mm, flow rate of 0.5 min/min, 0.08 MPa), was calibrated with a calibration solution in MEC buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). Fractions were monitored by recording the absorbance at 280 nm and the resulting relative mobility of the component protein plotted against the log of the respective molecular weights. The equation of the trendline is given by y = - 30.71x + 108.7, with a correlation coefficient of 0.991.
3.2.3 Recombinant expression and purification of full length oligopeptidase B from T. congolense, (TcOPB)
TcOPB has been previously cloned into the pET28-a bacterial expression vector and transformed into BL21 (DE3) E. coli cells by Lorelle Bizaaré (2008). One litre of 2 x YT liquid medium [1.6% (w/v) tryptone, 1% (w/v) yeast extract, 0.5% (w/v) NaCl containing 34 µg/ml kanamycin] was inoculated with a single recombinant pET-28a BL21 (DE3) colony. After incubation 37°C for 16 h in baffled flasks, the cells were pelleted by centrifugation (4 000 g, 10 min, 4°C). The pellet was resuspended in 1% (v/v) Triton X-100-PBS (20 ml), lysozyme added (1 mg/ml final concentration) and incubated at 37°C for 30 min. The cell suspension was frozen at -70°C for 1 h and subsequently thawed at RT. The cell suspension was sonicated four times for each 30 s respectively and the cellular debris was subsequently pelleted from the soluble protein lysate by centrifugation (5 000 g, 10 min, 4°C). The protein lysate was filtered through Whatman No. 1 filter paper and stored at -20°C. Samples of the supernatant and the pellet containing the soluble and insoluble fractions respectively were analysed by a 12.5%
reducing SDS-PAGE gel (Laemmli, 1970) and stained with Coomassie blue R-250 (as per section 2.2.6).
His-select® nickel affinity resin (1 ml) was placed in a 10 ml chromatography column, washed with 2 column volumes of dH2O and equilibrated with 5 column volumes of equilibration buffer (50 mM NaH2PO4, 0.5 M NaCl, 10 mM imidazole, pH 6.8).
Expression lysate (9 ml) was incubated with the resin for 4°C for 3 h and mixed using an end-over-end rotator. The unbound proteins were collected and the resin washed with 40 ml equilibration buffer until an absorbance value at 280 nm of 0.02 was reached. The bound proteins were eluted in 1 ml fractions with 15 ml elution buffer (50 mM NaH2PO4, 0.3 M NaCl, 50 mM imidazole, pH 8.0). The resin was regenerated with 2 column volumes of dH2O, 5 column volumes of 6 M guanidine hydrochloride, 3 column volumes of dH2O, and 3 column volumes of equilibration buffer and stored in 30% (v/v) ethanol at 4°C. Samples of the unbound and the eluted fractions were electrophoresed on a 12.5% reducing SDS-PAGE gel (Laemmli, 1970) and stained with Coomassie blue R-250 (as per section 2.2.7).
The fractions containing the purified TcOPB enzyme were pooled and dialysed against 3 changes of MEC buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) at 4°C and was subsequently concentrated against polyethylene glycol (PEG) Mr 20 000.
Molecular exclusion chromatography (MEC) was performed using a HiPrep™ 16/16 Sephacryl™ S300 HR resin (16 x 600 mm) with the ÄKTApurifier®. After equilibration with 180 ml MEC buffer, the protein sample (2 ml) was loaded onto the column. The resin was eluted at 0.08 kPa with a flow rate of 0.5 ml/min, with 1 ml fractions collected and the absorbance recorded at 280 nm. Samples at the corresponding elution volume of TcOPB were analysed by 12.5% reducing SDS-PAGE gel (Laemmli, 1970) and visualised using a silver stain (Blum et al., 1987). The resin was regenerated with 60 ml of a NaOH solution (0.2 M) and re-equilibrated with 180 ml MEC buffer.
The TcOPB containing fractions from the MEC purification were pooled and dialysed against 3 changes of 50 mM phosphate buffer, pH 7.2 at 4°C. The dialysed sample was concentrated using an Amicon® Ultra-15 centrifugal concentrator according to the manufacturer‟s instructions. The final concentration of TcOPB was determined using the BCA™ protein assay kit as per manufacturer‟s instructions (as per section 2.2.3).
3.2.4 Recombinant expression and purification of full length oligopeptidase B from T. vivax, (TvOPB), and pyroglutamyl peptidase from T. congolense, (TcPGP)
Both TcPGP and TvOPB were all previously cloned into pGEX4T-1 bacterial expression vectors and transformed into BL21 (DE3) E. coli cells by Laura Huson (2006) and Hérmogénes Mucache (2012) respectively. One hundred ml of 2 x YT liquid medium (containing 50 µg/ml ampicillin) was inoculated with a single recombinant pGEX4T-1 BL21 (DE3) colony and incubated at 37°C for 16 h in baffled flasks. The
overnight culture was diluted 1:100 with 900 ml fresh 2 x YT medium (containing 50 µg/ml ampicillin) and grown at 37°C in baffled flasks with agitation until an OD600 of 0.7 was reached. Expression was induced with IPTG (0.1 M) at a final concentration of 0.3 mM, at 37°C for 2 h with agitation. Ampicillin (50 μg/ml, 1 ml) was added at the start of induction as well as 2 h thereafter. The cells were pelleted by centrifugation (2 000 g, 10 min, 4°C) and resuspended in 1% (v/v) Triton X-100-PBS (20 ml). Lysozyme was added (1 mg/ml final concentration) and incubated at 37°C for 30 min. The cell suspension was frozen at -70°C for 1 h and subsequently thawed at RT. The cell suspension was sonicated four times for each 30 s respectively and the cellular debris was subsequently pelleted from the soluble protein lysate by centrifugation (5 000 g, 10 min, 4°C). The protein lysate was filtered through Whatman No. 1 filter paper and stored at -20°C. Samples of the supernatant and the pellet containing the soluble and insoluble fractions respectively were analysed by 12.5% reducing SDS-PAGE (Laemmli, 1970) and stained with Coomassie blue R-250 (as per section 2.2.6).
Glutathione agarose resin (1 ml) was placed in a 10 ml chromatography column, washed and equilibrated with 1% (v/v) Triton X-100-PBS (20 ml). Expression lysate (9 ml) was added to the resin and mixed at 4°C for 3 h using an end-over-end rotator.
The unbound proteins were collected and the resin washed with 1% (v/v) Triton X-100-PBS (50 ml) until an absorbance value at 280 nm of 0.02 was reached.
The resin was subsequently equilibrated with 20 ml thrombin cleavage buffer (20 mM Tris-HCl buffer, 150 mM NaCl, 2.5 mM CaCl2, pH 8.4) and resuspended in 0.8 ml thrombin cleavage buffer to which thrombin was added (2 U). The resin was mixed at 4°C for 16 h using an end-over-end rotator. Cleaved recombinant protein was eluted in 1 ml fractions with 9 ml thrombin cleavage buffer and the bound GST and uncleaved GST fusion protein being eluted with 10 ml reduced glutathione (10 mM in 50 mM Tris-HCl buffer, pH 8.0). The resin was regenerated with 5 column volumes of sodium borate buffer (200 mM in 500 mM NaCl, pH 8.0), 5 column volumes of sodium acetate buffer (100 mM in 500 mM NaCl, pH 4.0), 5 column volumes of dH2O and stored in 30% (v/v) ethanol at 4°C. Samples of each fraction were analysed by 12.5% reducing SDS-PAGE (Laemmli, 1970) and stained with Coomassie blue R-250 (as per section 2.2.7).
The fractions containing the enzyme were pooled and dialysed against 3 changes of dialysis buffer (50 mM phosphate buffer, pH 7.2) at 4°C and subsequently concentrated
against PEG 20 000. Protein concentration was determined using the BCA™ protein assay kit (as per section 2.2.3).
3.2.5 Recombinant expression and purification of the catalytic domain of vivapain from T. vivax, (TvCATL), full length congopain, (TcCATL
FL), and the catalytic domain of congopain, (TcCATL), from T. congolense
Pichia pastoris is a methylotropic yeast which has been utilised for high levels of expression of recombinant proteins which is regulated by the alcohol oxidase promoter (AOX, EC 1.1.3.13) (Cregg and Madden, 1988; Cereghino et al., 2002). TvCATL (catalytic domain of vivapain), TcCATLFL (full length congopain) and TcCATL (catalytic domain of congopain) have all be previously cloned into pPIC 9 yeast expression vectors and transformed into GS115 yeast cells by Perina Vather (2010), and Davita Pillay (2010) respectively. These constructs were expressed and purified in the same manner unless otherwise specified.
Glycerol stocks of TvCATL, TcCATLFL and TcCATL were streaked onto individual yeast extract peptone dextrose plates (YPD) [1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose, 1% (w/v) bacteriological agar containing 10 μg/ml tetracycline] and incubated at 30°C for 3 days. Fifty millilitres of YPD liquid medium [1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose containing 10 µg/ml tetracycline] was inoculated with a single colony and grown in baffled flasks at 30°C for 2 days with agitation. The YPD culture was added to 450 ml buffered media glycerol yeast (BMGY) [1% (w/v) yeast extract, 2% (w/v) peptone, 100 mM potassium phosphate buffer, pH 6.5, 1.34% (w/v) yeast nitrogen base without amino acids (YNB) containing 10 μg/ml tetracycline and 50 μg/ml ampicillin] and was subsequently grown at 30°C for a further 3 days with agitation. The yeast cells were pelleted by centrifugation (2 000 g, 10 min, 4°C) and resuspended in 500 ml buffered minimal media (BMM) [100 mM potassium phosphate buffer, pH 6.5, 1.34% (w/v) YNB, 0.0004% (w/v) biotin, 0.5% (v/v) methanol containing 10 μg/ml tetracycline and 50 μg/ml ampicillin]. The resulting cell suspension was transferred into baffled culture flasks and covered with 3 layers of sterile cheesecloth to facilitate aeration. Expression of each of the recombinant proteins occurred over 7 days at 30°C with agitation and the addition of 0.5% (v/v) methanol every 24 h.
The yeast cells were pelleted (2 000 g, 10 min, 4°C) and the supernatants, containing the expressed proteins, collected. Three phase partitioning (TPP), developed by Pike and Dennison (1989), was implemented as an initial concentration step whereby the
recombinantly expressed protein is precipitated using ammonium sulfate and tertiary-butanol. As a result, the tertiary-butanol increases the buoyancy of the precipitated protein which is found above the denser aqueous phase. Briefly, the yeast supernatant (500 ml) was filtered through Whatman No. 4 filter paper to which tertiary-butanol was added to a final concentration of 30% (v/v). Ammonium sulfate was added to a final concentration of 30% (w/v) and stirred until completely dissolved. A final concentration of 40% (w/v) ammonium sulfate was required for TvCATL. The resulting mixture was centrifuged (6 000 g, 10 min, 4°C) in a spin out rotor to achieve three separate phases. The upper tertiary-butanol and lower aqueous phases were removed leaving the protein precipitate. The protein precipitate was dissolved in the least volume of dialysis buffer and dialysed against 3 changes of dialysis buffer at 4°C and was subsequently concentrated against PEG 20 000.
Molecular exclusion chromatography (MEC) was performed using a HiPrep™ 16/16 Sephacryl™ S200 HR and S 300 HR resins (16 x 600 mm), where specified, with the ÄKTApurifier®. After equilibration with 180 ml MEC buffer, the protein sample (2 ml) was loaded onto the column. The resin was eluted at 0.08 kPa with a flow rate of 0.5 ml/min, with 1 ml fractions collected and the absorbance recorded at 280 nm.
Samples at the corresponding elution volume of the protein loaded, were analysed by 12.5% reducing SDS-PAGE (Laemmli, 1970) and visualised using a silver stain (Blum et al., 1987). The resin was regenerated with a 60 ml NaOH solution (0.2 M) and re-equilibrated with 180 ml MEC buffer.
The protein containing fractions from the MEC purification were pooled and dialysed against 3 changes of dialysis buffer, at 4°C and subsequently concentrated using Amicon® Ultra-15 centrifugal concentrators according to the manufacturer‟s instructions. Protein concentration was determined using the BCA™ protein assay kit (as per section 2.2.3).
3.2.6 Antibody production and ELISA optimisation
3.2.6.1 Coupling of the TcCATL N-terminal peptide to rabbit albumin via MBS
The TcCATL N-terminal peptide (4 mg) was dissolved in DMSO (150 µl) and reducing buffer [100 mM Tris-HCl buffer, pH 8.0, 10 mM Na2EDTA, 1% (w/v) SDS (350 µl)] was subsequently added. After the addition of DTT (10 mM, 500 μl), the mixture was incubated at 37°C for 1.5 h. The reduced peptide was separated from DTT using a
Sephadex G-10 column (15 x 110 mm, 0.6 ml/min), previously equilibrated with 50 ml MEC buffer [100 mM sodium phosphate buffer, pH 7.0, 0.02% (w/v) NaN3]. The column was eluted with 25 ml MEC buffer at a flow rate of 0.6 ml/min with 0.5 ml fractions being collected. A sample of each fraction (10 μl) was tested with 10 µl of Ellman‟s reagent (10 mg Ellman‟s reagent [5,5'-Dithio-bis-(2-nitrobenzoic acid)] in 2.5 ml Ellman‟s reagent buffer [100 mM Tris-HCl buffer, pH 8.0, 10 mM Na2EDTA, 1% (m/v) SDS]) with the first fractions to give a yellow reaction, i.e. the reduced peptide, being subsequently pooled (Kitagawa and Aikawa, 1976).
Rabbit albumin (2.794 mg) was dissolved in PBS (895 μl) to which MBS (102 μl) was subsequently added resulting in a 40:1 rabbit albumin to MBS molar ratio. The resulting solution was stirred at RT for 30 min. The MBS-rabbit albumin solution was loaded onto a Sephadex G-25 resin (15 x 130 mm) previously equilibrated with 50 ml chromatography buffer (100 mM NaH2PO4, pH 7.0). The resin was eluted at a flow rate of 10 ml/min and 1 ml fractions were collected. Elution was monitored by measuring the absorbance at 280 nm, with the activated rabbit albumin carrier being eluted in the first peak. The reduced peptide and activated rabbit albumin carrier fractions were combined and stirred at RT for 3 h. The solution was divided into four equal aliquots and stored at -20°C until immunisation.
3.2.6.2 Preparation of immunogen for the immunisation of chickens and IgY isolation from the egg yolk
As outlined in section 2.2.9.1, chickens were used to raise antibodies against the purified recombinant proteins, TcOPB, TvOPB, TcPGP, TvCATL, TcCATLFL and TcCATL. In addition to the recombinant proteins, the rabbit albumin coupled TcCATL N-terminal peptide was also used to raise antibodies. Three chickens for each of the recombinant proteins and two chickens for the TcCATL N-terminal peptide were immunised intramuscularly on either side of the breast bone. Each of the recombinant proteins (50 µg/ml, 1.5 ml) and the conjugated TcCATL N-terminal peptide (1.1 ml) were added to an equal volume of Freund‟s adjuvant and triturated to form a stable oil-in-water emulsion prior to immunisation.
3.2.6.3 ELISA evaluation of antibody production
The protocol was as outlined in section 2.2.9.2. Two buffers were used as the antigen coating diluent; PBS (100 mM Na2HPO4, 2 mM KH2PO4, 2.7 mM KCl and 137 mM NaCl, pH 7.4) and carbonate coating buffer (CCB) (50 mM carbonate buffer, pH 9.6).
3.2.7 Affinity purification of isolated IgY antibodies
3.2.7.1 Coupling of TcCATL N-terminal peptide to SulfoLink
®The TcCATL N-terminal peptide (5 mg) was dissolved in DMSO (100 µl) and 400 µl SulfoLink coupling buffer (50 mM Tris-HCl buffer, 5 mM Na2EDTA, pH 8.5) was added.
After the addition of DTT (10 mM, 500 μl), the mixture was incubated at 37°C for 1.5 h.
The reduced peptide was separated from DTT using a Sephadex G-10 column (15 x 110 mm), previously equilibrated with 50 ml MEC buffer (as per section 3.2.6.1).
The column was eluted with 25 ml MEC buffer at a flow rate of 0.6 ml/min and 0.5 ml fractions collected. A sample of each fraction (10 μl) was tested with 10 µl Ellman‟s reagent [10 mg Ellman‟s reagent in 2.5 ml Ellman‟s reagent buffer [100 mM Tris-HCl buffer, pH 8.0, 10 mM Na2EDTA, 1% (w/v) SDS)] with the first fractions to give a yellow reaction, i.e. reduced peptide, being pooled.
SulfoLink® resin (1 ml) was placed in a 10 ml chromatography column and equilibrated with 6 column volumes of general buffer to which the reduced peptide was added. The resin was mixed for 15 min at RT using an end-over-end rotator. The resin was washed with 16 column volumes of wash buffer (1 M NaCl) followed by 2 column volumes of IgY buffer and stored at 4°C until use.
3.2.7.2 Coupling of TcCATL
FLto AminoLink
®Purified TcCATLFL was coupled to AminoLink® coupling gel for the affinity purification of antibodies produced against the recombinant protein. AminoLink® coupling gel (1 ml) was placed in a 10 ml chromatography column and equilibrated with 6 column volumes of Aminolink coupling buffer [100 mM NaH2PO4, 0.05% (w/v) NaN3, pH 7.4]. Purified TcCATLFL (5 mg/ml, 1 ml) was diluted in coupling buffer (3 ml) and, along with 5 M NaCNBH3 in 1 M NaOH (50 μl), was added to the resin. The resin was mixed for 2 h at RT using an end-over-end rotator and allowed to settle for 16 h at 4°C. The unbound fraction was collected, the column was washed with 4 ml coupling buffer and subsequently with 4 ml quenching buffer (1 M Tris-HCl buffer, pH 7.4). To the resin, 2 ml quenching buffer and 5 M NaCNBH3 in 1 M NaOH (50 μl) was added and mixed for 2 h at RT using an end over end rotator. The resin was subsequently washed with 25 ml wash buffer, until the absorbance at 280 nm reached baseline, followed by 6 ml IgY buffer and stored at 4°C until use.
The isolated IgY antibodies, from section 3.2.6.2, were pooled and filtered through Whatman No. 1 filter paper. The affinity columns were equilibrated with 5 column
volumes of wash buffer [100 mM NaH2PO4, 0.05% (w/v) NaN3, pH 6.5] and each pool cycled through the column for 16 h at RT at a flow rate of 0.6 ml/min. The unbound IgY was collected and the resin was subsequently washed with 10 column volumes of wash buffer until an absorbance value at 280 nm of 0.02 was obtained. The bound IgY, specific to the antigen in question, was eluted with elution buffer [100 mM glycine-HCl, 0.02% (w/v) NaN3, pH 2.8] and 900 μl fractions collected into microcentrifuge tubes containing 100 µl neutralisation buffer [1 M NaH2PO4, 0.02% (w/v) NaN3, pH 8.5] and each was immediately mixed by inversion. Elution of the specific antibodies was monitored spectrophotometrically at 280 nm using 900 μl of elution buffer and 100 μl neutralisation buffer as the blank. The affinity purified IgY-containing fractions were pooled, with 10% (w/v) NaN3 being subsequently added to a final concentration of 0.1% (w/v) before storing at 4°C. The affinity columns were regenerated using 12 column volumes of wash buffer and stored at 4°C until use.
3.2.8 Western blot of recombinantly purified proteins with their respective IgY antibodies raised in chickens
Western blotting was performed as outlined in section 2.2.6 using the Pierce™ ECL western blotting substrate.