3.3 Results
3.3.2 Recombinant expression and purification of full length oligopeptidase B from
peptidase from T. congolense, (TcPGP)
3.3.2.1 Full length oligopeptidase B from T. vivax, (TvOPB)
The expression of recombinant TvOPB with the glutathione S-transferase (GST) fusion tag at 106 kDa was achieved as seen in Fig. 3.5, panel A. The expression lysates were subjected to glutathione agarose based affinity chromatography making use of the GST fusion tag which was introduced to TvOPB by the pGEX4T-1 expression vector.
Purification was successful with cleaved TvOPB being eluted at 80 kDa in fractions 1 to 8, along with uncleaved TvOPB fusion protein, cleaved TvOPB and cleaved GST at 105, 80 and 25 kDa, respectively, in the reduced glutathione fractions (Fig. 3.5, panel B). An increased concentration of thrombin and an increased number of fractions collected before reduced glutathione elution commenced would have resulted in a
higher yield of pure cleaved TvOPB. The purified TvOPB in fractions 1 to 8 were pooled, concentrated and used for antibody production.
Figure 3.5: Analysis of recombinantly expressed TvOPB by IPTG induction and the subsequent affinity purification. (A) Samples of the expression of TvOPB by IPTG induction were analysed by a 10% reducing SDS-PAGE gel. (B) Samples of the glutathione S-transferase affinity chromatography purification of expression supernatants were analysed by a 12.5% reducing SDS-PAGE gel. Proteins were stained with Coomassie blue R-250. Ni: non-induced control sample.
3.3.2.2 Pyroglutamyl peptidase from T. congolense, (TcPGP)
The expression of recombinant TcPGP with the GST fusion tag at 51 kDa was achieved as is shown in Fig. 3.6, panel A. The expression lysates were subjected to glutathione agarose based affinity chromatography making use of the GST fusion tag, introduced to the TcPGP protein by the pGEX4T-1 expression vector. Purification was successful, with pure cleaved TcPGP at 28 kDa eluting in fractions 1 to 10, and the uncleaved TcPGP fusion protein and cleaved GST at 53 and 25 kDa, respectively, in the reduced glutathione fractions (Fig. 3.6, panel B). The purified TcPGP in fractions 1 to 10 were pooled, concentrated and used for antibody production.
Figure 3.6: Analysis of recombinantly expressed TcPGP by IPTG induction and the subsequent affinity purification. (A) Samples of the expression of TcPGP by IPTG induction were analysed by a 10% reducing SDS-PAGE gel. (B) Samples of the glutathione S-transferase affinity chromatography purification of expression supernatants were analysed by a 12.5% reducing SDS-PAGE gel. Proteins were stained with Coomassie blue R-250. Non-induced control sample.
3.3.3 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
3.3.3.1 Catalytic domain of vivapain from T. vivax, (TvCATL)
The expression of recombinant TvCATL, in its glycosylated and non-glycosylated forms, at 29 and 33 kDa was achieved as seen in Fig. 3.7, panel A. The expression supernatants were subsequently subjected to TPP purification. Purification was successful with two bands at 29 and 33 kDa being visualised (Fig. 3.7, panel B). The protein sample after TPP was diluted in a larger volume of dialysis buffer than what was needed and resulted in a very dilute sample. Due to the high dilution of the protein sample after TPP, any higher or lower molecular weight contaminating proteins were not visible and thus required further purification by MEC (Fig. 3.8).
Figure 3.7: Analysis of the expression of recombinant TvCATL in Pichia pastoris and purification on a 12.5% reducing SDS-PAGE gel. (A) Expression of TvCATL after 7 days in BMM. (B) The expression supernatant was subjected to TPP with 40% (w/v) ammonium sulfate to purify TvCATL. Proteins were stained with Coomassie blue R-250.
TvCATL is glycosylated in the yeast system and thus two peaks following purification by MEC should be expected (Vather, 2010; Jackson, 2011). According to the calibration curve for the S200 HR molecular exclusion resin (Fig. 3.2), TvCATL was predicted to have retention volumes of approximately 65.9 and 63.98 ml for the 29 and 33 kDa TvCATL proteins respectively. Three peaks with retention volumes of 35.82, 59.13 and 72.05 ml were the result of the molecular exclusion of TvCATL (Fig. 3.8, panel A). As shown in Fig. 3.8, panel B, pure glycosylated and de-glycosylated TvCATL were eluted around a retention volume of 72.13 ml in fractions 64 to 85 and required no further purification. The peaks at the retention volumes of 35.82 and 59.13 ml were attributed to higher molecular weight contaminating proteins. Fractions 65 to 85 containing purified glycosylated and non-glycosylated TvCATL were pooled, dialysed and used for antibody production.
Figure 3.8: Purification of recombinant TvCATL using a HiPrep™ 16/16 Sephacryl™ S200 HR molecular exclusion resin. (A) Elution profile of TvCATL on a HiPrep™ 16/16 Sephacryl™ S200 HR resin (16 x 600 mm, flow rate 0.5 min/min, 0.08MPa), previously equilibrated with MEC buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and eluted using MEC buffer. Fractions were monitored by recording the absorbance at 280 nm. (B) Samples of the eluted fractions were analysed by a 12.5% reducing SDS-PAGE gel. Proteins were visualised using silver staining.
3.3.3.2 Full length congopain from T. congolense, (TcCATL
FL)
The expression of recombinant TcCATLFL at 40 kDa was achieved as shown in Fig. 3.9, panel A, and the expression supernatants subjected to TPP purification.
Purification was successful, yielding a high concentration of TcCATLFL at 40 kDa along with lower and higher molecular weight contaminating proteins which required further purification by MEC (Fig. 3.9, panel B).
Figure 3.9: Analysis of the expression of recombinant TcCATLFL in Pichia pastoris and purification on a 10% reducing SDS-PAGE gel. (A) Expression of TcCATLFL after 7 days in BMM. (B) The expression supernatant was subjected to TPP with 30% (w/v) ammonium sulfate to purify TcCATLFL. Proteins were stained with Coomassie blue R-250.
According to the calibration curve for the S200 HR molecular exclusion resin (Fig. 3.2), TcCATLFL was predicted to have a retention volume of approximately 61.1 ml. Three peaks with retention volumes of 36.41, 50.50 and 61.90 ml were the result of the MEC of TcCATLFL (Fig. 3.10, panel A). From Fig. 3.10, panel B, TcCATLFL is present in fractions 60 to 96. A protein of a lower molecular weight than TcCATLFL was present in fractions 65 to 72 along with TcCATLFL. High levels of TcCATLFL expression was achieved using the Pichia pastoris system and thus was present throughout the collected fractions during MEC along with a lower molecular weight protein eluted in fractions 65 to 72. Thus, one peak could not be accurately chosen to represent the retention volume of TcCATLFL, however the last peak at 61.90 ml compares favourably to the predicted elution volume of TcCATLFL. Fractions 65 to 72 underwent further MEC purification to remove the lower molecular weight protein from TcCATLFL. Fractions 58 to 81 and 91 to 97 containing pure TcCATLFL were pooled, dialysed and used for antibody production.
Figure 3.10: Purification of recombinant TcCATLFL using a HiPrep™ 16/16 Sephacryl™ S200 HR molecular exclusion resin. (A) Elution profile of TcCATLFL on a HiPrep™ 16/16 Sephacryl™ S200 HR resin (16 x 600 mm, flow rate 0.5 min/min, 0.08 MPa), previously equilibrated with MEC buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and eluted using MEC buffer. Fractions were monitored by recording the absorbance at 280 nm. (B) Samples of the eluted fractions were analysed by a 12.5% reducing SDS-PAGE gel. Proteins were visualised using silver staining.
3.3.3.3 Catalytic domain of congopain from T. congolense, (TcCATL)
The expression of recombinant TcCATL at 27 kDa was achieved as seen in Fig. 3.11, panel A, and the expression supernatants subjected to TPP purification. Purification was successful, yielding a high concentration of TcCATL at 27 kDa along with higher molecular weight contaminating proteins which required further purification by MEC (Fig. 3.11, panel B).
Figure 3.11: Analysis of the expression of recombinant TcCATL in Pichia pastoris and purification on a 10% reducing SDS-PAGE gel. (A) Expression of TcCATL after 7 days in BMM. (B) The expression supernatant was subjected to TPP with 30% (w/v) ammonium sulfate to purify TcCATL. Proteins were stained with Coomassie blue R-250.
According to the calibration curve for the S200 HR molecular exclusion resin (Fig. 3.2), TcCATL was predicted to have a retention volume of approximately 66.99 ml. Two peaks with retention volumes of 35.99 and 68.61 ml were present following the molecular exclusion of TcCATL (Fig. 3.12, panel A). Pure TcCATL, present in fractions 64 to 76, had a retention volume of 68.61 ml and compared favourably with the predicted value (Fig. 3.12, panel B). The peak with a retention volume of 36.32 ml was attributed to the higher molecular weight contaminating proteins as seen in Fig. 3.11, panel B. Fractions 64 to 76 containing pure TcCATL were pooled, dialysed and used for antibody production.
Figure 3.12: Purification of recombinant TcCATL using a HiPrep™ 16/16 Sephacryl™ S200 HR molecular exclusion resin. (A) Elution profile of TcCATL on a HiPrep™ 16/16 Sephacryl™ S200 HR resin (16 x 600 mm, flow rate 0.5 min/min, 0.08 MPa), previously equilibrated with MEC buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) and eluted using MEC buffer. Fractions were monitored by recording the absorbance at 280 nm. (B) Samples of the eluted fractions were analysed by a 12.5% reducing SDS-PAGE gel. Proteins were visualised using silver staining.