2. Expression and Purification of Par-
2.6. Purification of Par-4(1-332)
2.6.1. Purification of Par-4(1-332)WT from HpMal-c2P a) Materials and Methods
For conciseness, purification steps are described once in detail as a general protocol. For later protocols only differences to this general protocol will be described. Unless stated otherwise all Par-4 purifications were performed on ice or at 4 ºC. All purification methods are described for a culture volume of one litre.
The cell pellet was resuspended in 20 ml PBS and 100 µl of the protease inhibitor cocktail
were added. The cell suspension was passed three times through a French Press. The cell lysate was clarified by centrifugation for 30 min at 14,000 g (Rotor FA12.94 Highconic). The
supernatant was passed through a 0.8 µm syringe filter and the filtrate rotated for 3 h with 2 ml
(bed-volume) Chelating Sepharose, which had been charged with NiCl2 and equilibrated to PBS. The Ni-NTA column was washed successively with 10 ml PBS, 15 ml PBS with 1 M NaCl and 10 ml PBS. For cleavage of the Par-4 fusion protein, the TCEP concentration was adjusted to 0.3 mM, HMBP-3Cpro was added and the Ni-NTA column was rotated overnight at 4 ºC. In case of incomplete cleavage of the Par-4 fusion protein more HMBP-3Cpro was added and the cleavage reaction continued till more than 90% of the fusion protein was cleaved. Cleaved Par-4 (1-332)WT was eluted from the column with 10 ml PBS. Par-4(1-332)WT containing fractions were pooled and concentrated to 2 ml using a Vivaspin 20 device (MWCO = 10 kDa) at 3,000 g (Rotor TTH400). To remove traces of residual MBP, the concentrate was applied to 2 ml Amylose resin and the flow-through collected.
b) Results
Par-4(1-332)WT in HpMal-c2P was expressed in an unlabelled form with a final OD600 = 4 after overnight expression. The Par-4 fusion protein was mostly soluble, but 40-50% remained in the insoluble fraction after clarification of the cell lysate. The IMAC purified Par-4 fusion protein was 70-80% pure and was cleaved on the column with HMBP-3Cpro. Adding TCEP to a concentration of 0.3 mM improved the cleavage performance of HMBP-3Cpro and nearly 90% of the Par-4 fusion protein was cleaved. Due to the same electrophoretic mobility of MBP and Par-4(1-332)WT, only one band corresponding to a 45 kDa protein is visible by SDS-PAGE. Amylose resin purification showed that all eluted protein bound to the Amylose resin, indicating
that only MBP eluted from the Ni2+-Sepharose column. Cleaving the Par-4 fusion protein in
solution, resulted in protein precipitation, suggesting that Par-4(1-332)WT without the MBP tag is mostly insoluble in solution. Hence no Par-4(1-332)WT could be purified with this method.
2.6.2. Purification of Par-4(1-332)WT from pProEX-HTb a) Materials and Methods
The cell pellet was resuspended in 20 ml 100 mM NaCl, 25 mM imidazole and 50 mM
Na2HPO4/NaH2PO4, pH 8.0 and 100 µl of the protease inhibitor cocktail were added. Cell lysis
and incubation with Chelating Sepharose are described in Chapter 2.6.1. The Ni-NTA column was washed successively with 10 ml lysis buffer and 5 ml lysis buffer with 50 mM imidazole. The Par-4 fusion protein was eluted with 10 ml 100 mM NaCl, 250 mM imidazole and 50 mM Na2HPO4/NaH2PO4, pH 8.0. Par-4 fusion protein containing fractions were pooled and dialysed overnight against 500 ml 100 mM NaCl, 25 mM imidazole, 0.3 mM TCEP and 20 mM Tris, pH 8.0. The Par-4 fusion protein was cleaved for 4 h at room temperature with rTEV (Chapter 2.2.2.).
b) Results
Par-4(1-332)WT in pProEX-HTb was expressed in an unlabelled form with a final OD600 = 5 after overnight expression. A protein of the correct size (~45 kDa), as estimated by SDS-PAGE, was purified by IMAC. Dialysis of this protein against rTEV cleavage buffer (100 mM NaCl, 25 mM Imidazole, 0.3 mM TCEP and 20 mM Tris, pH 8.0) resulted in protein precipitation. Cleavage of the remaining soluble protein with rTEV showed no cleavage activity, suggesting that the isolated protein is a bacterial protein without rTEV cleavage site. Optimising induction of protein expression with various IPTG concentrations (final concentrations 0-0.5 mM) showed
no upregulated protein relative to the non-induced cell culture, indicating that Par-4(1-332)WT is not expressed from pProEX-HTb.
2.6.3. Purification of Par-4(1-332)G40G from pET32a a) Materials and Methods
Cell lysis and IMAC purification are described in Chapter 2.6.2. Par-4 fusion protein containing fractions were pooled and dialysed overnight against 500 ml 150 mM NaCl, 0.5 mM EDTA, 0.3 mM TCEP and 50 mM Tris, pH 7.5. The Par-4 fusion protein was cleaved overnight with 1 mg Thrombin.
b) Results
Par-4(1-332)G40G in pET32a was expressed in an unlabelled form with a final OD600 = 4 after overnight expression. A 60 kDa protein, as estimated by SDS-PAGE, was isolated by IMAC and was more than 90% pure after elution. As described in Chapter 2.6.1, over 50% of the Par-4 fusion protein remained in the insoluble fraction after clarification of the cell lysate. Cleavage of the Par-4 fusion protein with Thrombin resulted in the degradation of the Par-4 fusion protein as was indicated by SDS-PAGE with protein bands below 20 kDa.
2.6.4. Native purification of Par-4(1-332)G40G from pET32TEV a) Materials and Methods
Cell lysis and IMAC purification are described in Chapter 2.6.2. Par-4 fusion protein containing fractions were pooled and dialysed overnight against 500 ml 100 mM NaCl, 25 mM imidazole, 0.3 mM TCEP and 20 mM Tris, pH 8.0.
b) Results
Par-4(1-332)G40G in pET32TEV was expressed in an unlabelled form with a final OD600 = 4 after overnight expression. Using a standard lysis buffer with 100 mM NaCl, approximately 90% of the Par-4 fusion protein remained in the insoluble fraction after clarification of the cell lysate. Lysis conditions were optimised with six different buffers, testing two NaCl concentrations (250 and 500 mM) at three different pH values (pH 6.0, 7.0 and 8.0). The imidazole and Na2HPO4/ NaH2PO4 concentration were the same in all six lysis buffers. Using a lysis buffer containing 500 mM NaCl at pH 8.0, almost 90% of the Par-4 fusion protein remained in the soluble fraction after clarification of the cell lysate. Note that at pH 6.0 the Par-4 fusion protein was virtually
insoluble, with intermediate solubility at pH 7.0. The IMAC purified Par-4 fusion protein was ~80% pure after elution and had an apparent MW of 60 kDa as judged by SDS-PAGE (Fig. 2.1, lane 7). Dialysis against rTEV cleavage buffer resulted in an almost complete precipitation of the Par-4 fusion protein. Hence, no Par-4(1-332)G40G could be purified by this method.
2.6.5. Denaturing purification of Par-4(1-332)G40G from pET32TEV −
Purification of rrPar-4FL a) Materials and Methods
Unless stated otherwise, all steps of the denaturing purification were carried out at room temperature. The cell pellet was resuspended in 20 ml 8 M urea, 1 M NaCl, 25 mM imidazole,
100 mM NaH2PO4 and 10 mM Tris, pH 8.0 and 100 µl of the protease inhibitor cocktail were
added. The cell suspension was passed three times through a French Press. The cell lysate was clarified by centrifugation for 30 min at 14,000 g and 4 ºC (Rotor FA12.94 Highconic). The
supernatant was passed through a 0.8 µm syringe filter and the filtrate rotated for 3 h with 2 ml
(bed-volume) of Chelating Sepharose, which had been charged with NiCl2 and equilibrated to the lysis buffer. The Ni-NTA column was washed successively with 10 ml lysis buffer and 5 ml lysis buffer with 50 mM imidazole. The Par-4 fusion protein was eluted with 10 ml 8 M urea, 500 mM NaCl, 250 mM imidazole, 100 mM NaH2PO4 and 10 mM Tris, pH 8.0. Par-4 fusion protein containing fractions were pooled and dialysed for 6 h against 500 ml 4 M urea, 250 mM NaCl, and 50 mM Tris, pH 8.0, followed by an overnight dialysis against 500 ml cleavage buffer, containing 1 M urea, 100 mM NaCl, 25 mM imidazole, 0.3 mM TCEP and 50 mM Tris, pH 8.0. The Par-4 fusion protein was cleaved for 6 h with rTEV. After more than 90% of the Par-4 fusion protein was cleaved, the cleavage reaction was applied again to the Ni-NTA column, which had been equilibrated to the cleavage buffer. The flow through was collected and the Ni-NTA column washed with 5 ml cleavage buffer. Par-4(1-332)G40G containing fractions were pooled and concentrated to 2 ml using a Vivaspin 20 device (MWCO = 10 kDa) at 3,000 g and 25 ºC (Rotor TTH400).
Par-4(1-332)G40G was further purified either by ion-exchange chromatography (IEC) or RP- HPLC. Ion-exchange chromatography was performed using a HiTrap Q FF column connected to the BioLogic DuoFlow System (BioRad, Hercules, USA). Par-4(1-332)G40G was purified on a linear gradient of 0-60% high salt buffer over 20 min. The low salt buffer contained 1 M urea, 50 mM NaCl and 20 mM Tris, pH 7.5; the high salt buffer contained 1 M urea, 1 M NaCl and 20 mM Tris, pH 7.5. Par-4 was purified at 4 ºC with a flow rate of 0.8 ml/min. Par-4(1-332)
G40G containing fractions that were free of impurities, were pooled and concentrated using a Vivaspin 20 device (MWCO = 10 kDa) at 3,000 g and 4 ºC (Rotor TTH400). Buffer exchange was achieved by successive 1:1 dilution with 20 mM NaCl, 10 mM Tris, pH 7.0 and subsequent re-concentration. After 8 steps the buffer exchange was to 99.6% complete.
RP-HPLC was performed with a Delta Pak C18-300Å column connected to an Ultimate 3000 HPLC (Dionex Corporation, Sunnyvale, USA). Par-4(1-332)G40G was purified at 25 ºC with a flow rate of 1.5 ml/min using a linear water-acetonitrile gradient of 20-45% over 20 min in the
presence of 0.1% TFA (v/v). Par-4(1-332)G40G containing fractions that were free of impurities
were lyophilised and then resolubilised in 20 mM NaCl, 10 mM Tris, pH 7.0. The protein
concentration was determined by A280 and A205 absorbance measurements using the extinction
coefficient 13,075 M-1cm-1 and the relationship described by Scopes [190].
Figure 2.1− Denaturing purification of rrPar-4FL. A typical purification of Par-4 by IMAC is shown for rrPar-4FL using a denaturing purification protocol. Similar results are obtained for other Par-4 constructs using native protocols. All fractions were analysed by 11% SDS-PAGE. Expression of the thioredoxin-Par-4(1-332)G40G fusion protein is evident from the comparison of (1) non-induced and (2) induced E. coli cells. Note that the size of the Par-4 proteins as judged by SDS-PAGE is greater than what is expected from the amino acid sequence (66 kDa relative to 51 kDa for the fusion protein and 46 kDa relative to 36 kDa for rrPar-4FL). The cell lysate was clarified by centrifugation (3) and applied to a Ni- NTA column. After 3 h incubation the flow-through (4) was collected and the column washed successively with buffers containing 25 mM (5) and 50 mM (6) imidazole. The fusion protein was eluted from the column with 250 mM imidazole (7,8) and the Ni-NTA column assessed for remaining fusion protein (9). The fusion protein was dialysed against cleavage buffer containing 1 M urea (10) and incubated for 6 h with rTEV protease at room temperature (11). The cleavage reaction was applied again to a Ni-NTA column to remove the thioredoxin tag. After 3 h incubation the flow-through (12) was collected and the column washed with cleavage buffers (13,14). The Ni-NTA column was assessed for remaining rrPar-4FL (15). rrPar-4FL was then further purified by RP-HPLC (data not shown). Lanes with molecular weight (MW) markers are labelled with (M). Approximate molecular weights are shown on the left edge. 200 kDa 116 kDa 97 kDa 66 kDa 45 kDa 31 kDa 21 kDa M 1 2 3 4 5 6 7 8 9 M 10 11 M 12 13 14 15
b) Results
Using a denaturing purification protocol almost all Par-4 fusion protein remained in the soluble fraction after clarification of the cell lysate. The IMAC purified Par-4 fusion protein was 70-80% pure after elution. Reducing the urea concentration for the cleavage reaction resulted in precipitation of ~10% of the Par-4 fusion protein. The cleaved Par-4(1-332)G40G after the second IMAC purification was ~90% pure (Fig. 2.1, lane 12). Buffer exchange of the IEC- purified Par-4(1-332)G40G with a Vivaspin 20 device resulted in a complete loss of Par-4 (1-332) due to precipitation. Alternatively, dialysing the IEC-purified Par-4(1-332)G40G and then concentrating it, also lead to a complete loss due to precipitation.
Using RP-HPLC for final purification, improved the purity of Par-4(1-332)G40G (rrPar-4FL) to > 98% as judged by mass spectrometry (MS, Centre for Protein Research, University of Otago, Dunedin, New Zealand). Resolubilising the lyophilised rrPar-4FL in 20 mM NaCl, 10 mM Tris, pH 7.0 gave a concentrated (0.43 mM) soluble protein. Storage of this solution at 4 ºC resulted in protein precipitation after a few days. The protein yield using the denaturing purification protocol and RP-HPLC was ~20 mg rrPar-4FL per litre cell culture. Par-4(1-332) G40G purified by this protocol (denaturing purification, RP-HPLC) will be denoted as
rrPar-4FL throughout the remainder of this text.