CHAPTER 2 MATERIALS AND METHODS
2.6 Recombinant protein techniques
This section describes all techniques that were employed for the expression of recombinant PyBCCP from pT-PyBCCP and pGX-PyBCCP (pET-28a and pGEX-4T-1 constructs respectively). Biotinylation of rabbit albumin as a control for the biotinylation study is also described. SDS-PAGE analysis and Western blotting techniques had been described in Sections 2.3.1 and 2.3.2, respectively.
2.6.1 Expression of His6-PyBCCP and GST-PyBCCP 2.6.1.1 Materials
Cell resuspension buffer [0.02 M Tris-HCl, 0.25 M NaCl, 0.1% (w/v) NaN3, 1 mM DTT, 5%
(v/v) glycerol, 0.5 mM PMSF, pH 7]. Tris (0.24 g), NaCl (1.5 g), PMSF (8.7 mg), NaN3
(0.1 g), DTT (15 mg) and glycerol (5 ml) were completely dissolved in 85 ml of dH2O, pH adjusted to 8 with HCl and volume made up to 100 ml with dH2O.
2.6.1.2 Method
Expression of His6-PyBCCP
Three-way streak of cells containing the recombinant plasmid (pT-PyBCCP in Rosetta (DE3) pLysS) was made on 2×YT-agar plate supplemented with kanamycin (34 µg/ml) and chloramphenicol (30 µg/ml) and incubated (37ºC, 16 h). A single colony was used to inoculate 2×YT-media (5 ml) supplemented with kanamycin and chloramphenicol (2×YT- kan-cam) and the culture was incubated (37ºC, 16 h). The culture was diluted 1/100 with fresh 2×YT-kan-cam and incubated at 37ºC until the culture had an OD of 0.6 (at 600 nm) after which the culture was induced with or without IPTG (0.5 mM) and incubated (37ºC, 5 h or 18ºC, 16 h). Cells were harvested by centrifugation (3500 g, 15 min, 4ºC) and suspended in 1/100th of the culture volume with cell resuspension buffer. The cell suspension was subjected to three freeze-thaw cycles between 37ºC and –196ºC (liquid nitrogen for 2 min) and further disrupted by sonication on ice (10 cycles, 5 sec/burst, 1 min between bursts). The total cell lysate was analysed for expression by SDS-PAGE and Western blotting using monoclonal anti-His-tag antibody.
Expression of GST-PyBCCP
Three-way streak of cells containing the recombinant plasmid (pGX-PyBCCP in BL21) was made on 2×YT-agar plate supplemented with ampicillin (100 µg/ml) and incubated (37ºC, 16 h). A single colony was used to inoculate 2×YT-media (5 ml) supplemented with ampicillin 2×YT-amp) and the culture was incubated (37ºC, 16h). The cells were washed 2×
(1000 g, 5 min, RT) with fresh media to reduce the effect of β-lactamase secreted into the media by the cells. The culture was diluted 1/100 with fresh 2×YT-amp and incubated at 37ºC until OD600 nm of the culture was approximately 0.6 after which the culture was induced with or without IPTG (0.5 mM) and incubated (37ºC, 5 h). The culture was continuously supplemented with ampicillin (100 µg/ml) every hour during expression. For prolonged expression (18ºC, 16 h), the 2×YT media was supplemented with carbenicillin (100 µg/ml).
Cells were harvested by centrifugation (3500 g, 15 min, 4ºC) and suspended in 1/100th of the culture volume with cell resuspension buffer containing egg white lysozyme (1 mg/ml) and incubated (37ºC, 20 min). The cell suspension was subjected to three cycles of freeze thaw between 37ºC and –196ºC (liquid nitrogen for 2 min) and further disrupted by sonication on ice (10 cycles, 5 sec/burst, 1 min between bursts). The total cell lysate was analysed for expression by SDS-PAGE and Western blotting using monoclonal anti-GST antibody.
2.6.2 Purification and refolding of His6-PyBCCP 2.6.2.1 Materials
TALON® metal affinity resin (1.5 ml matrix volume).
Pellet wash buffer [2 M Urea, 0.02 M Tris-HCl, 2.5% (v/v) Triton X-100, pH 8]. Urea (60 g), Tris (1.2 g) and Triton X-100 (12.5 ml) were completely dissolved in 350 ml of dH2O, pH adjusted to 8 with HCl and volume made up to 500 ml with dH2O. The buffer was stored at RT.
Solubilisation buffer [6 M guanidine-HCl, 0.02 M Tris-HCl, 0.25 M NaCl, 0.02 M imidazole, pH 8]. Guanidine-HCl (57.3 g), Tris (0.24 g), NaCl (1.5 g), and imidazole (1.4 g) were completely dissolved in ~ 25 ml of dH2O, pH adjusted to 8 with HCl and volume made up to 100 ml with dH2O. The buffer was stored at RT.
Wash buffer 1 [8 M urea, 0.02 M Tris-HCl, 0.5 M NaCl, pH 8]. Urea (24 g), Tris (0.12 g) and NaCl (1.45 g) were completely dissolved in ~ 10 ml of dH2O, pH adjusted to 8 with HCl and volume made up to 50 ml with dH2O. The buffer was stored at RT.
Wash buffer 2 [8 M urea, 0.02 M Tris-HCl, 0.03 M imidazole, pH 8.0]. Urea (24 g), Tris (0.12 g) and imidazole (0.1 g) were completely dissolved in ~ 10 ml of dH2O, pH adjusted to 8 with HCl and volume made up to 50 ml with dH2O. The buffer was stored at RT.
Elution buffer [8 M urea, 0.02 M Tris-HCl, 1 M imidazole, pH 8]. Urea (24 g), Tris (0.12 g) and imidazole (3.4 g) were completely dissolved in ~ 10 ml of dH2O, pH adjusted to 8 with HCl and volume made up to 50 ml with dH2O. The buffer was stored at RT.
Dialysis buffer 1 [4 M urea, 0.02 M Tris-HCl, 0.25 M L-arginine, 5% (v/v) glycerol, pH 8].
Urea (120 g), Tris (1.21 g), L-arginine (21.8 g) and glycerol (25 ml) were completely dissolved in 250 ml of dH2O, pH adjusted to 8 with HCl and volume made up to 500 ml with dH2O.
Dialysis buffer 2 [2 M urea, 0.02 M Tris-HCl, 0.25 M L-arginine, 10% (v/v) glycerol, pH 8].
Urea (120 g), Tris (2.4 g), L-arginine (43.6 g) and glycerol (100 ml) were completely
dissolved in 850 ml of dH2O, pH adjusted to 8 with HCl and volume made up to 1 L with dH2O.
Dialysis buffer 3 [1 M urea, 0.02 M Tris-HCl, 0.25 M L-arginine, 15% (v/v) glycerol, pH 8].
Urea (60 g), Tris (2.4 g), L-arginine (43.6 g) and glycerol (150 ml) were completely dissolved in 750 ml of dH2O, pH adjusted to 8 with HCl and volume made up to 1 L with dH2O.
Dialysis buffer 4 [0.5 M urea, 0.02 M Tris-HCl, 0.25 M L-arginine, 20% (v/v) glycerol, pH 8]. Urea (30 g), Tris (2.4 g), L-arginine (43.6 g) and glycerol (200 ml) were completely dissolved in 650 ml of dH2O, pH adjusted to 8.0 with HCl and volume made up to 1 L with dH2O.
Equilibration buffer [0.02 M Tris-HCl, 0.25 M L-arginine, 0.5 mM PMSF, 0.1% (w/v) NaN3
20% (v/v) glycerol, pH 8]. Tris (2.4 g), L-arginine (43.6 g), PMSF (87 mg), NaN3 (1 g) and glycerol (200 ml) were completely dissolved in 650 ml of dH2O, pH adjusted to 8 with HCl and volume made up to 1 L with dH2O.
2.6.2.2 Method
Purification of insoluble recombinant His6-PyBCCP
TALON resin slurry (3 ml) containing ~ 1.5 ml of resin was placed into a BioRad affinity column washed with 10 column volumes (15 ml) of Solubilisation buffer. Insoluble protein pellet obtained from large scale expression (5 L) was washed 3 times with pellet wash buffer (12 000 g, 30 min, 4ºC). The protein pellet was dissolved in 50 ml of Solubilisation buffer and centrifuged (12 000 g, 30 min, 4ºC) to remove insoluble materials that may block the affinity column. The protein sample was continuously circulated over the affinity matrix (16 h, RT, 0.25 ml/min) in a reverse direction. The matrix was washed with 10 column volume of the following in this order: Solubilisation buffer, wash buffer 1 and wash buffer 2. Bound proteins were eluted from the matrix with the elution buffer (0.25 ml/min, 1 ml/fraction, 15 ml) and A280 nm of each fraction was recorded. Fractions with A280 nm > 0.5 were pooled and the purification profile was analysed by SDS-PAGE (Section 2.3.1).
Refolding of recombinant His6-PyBCCP
Dialysis membrane (10 kD MWCO) was washed; the eluted protein sample (2 ml) was placed in the dialysis bag and sealed. The protein was dialysed by continuous stirring against the buffers as illustrated in Table 2.7.
Table 2.7 Dialysis schedule for refolding of His6-PyBCCP
Buffer Vol
(ml)
Time (h)
Temp Number of changes
[Urea]
(M)
Estimated [urea] (mM) at equilibrium
Dialysis buffer 1 250 6 RT 2 4.0 32.0
Dialysis buffer 2 1000 18 RT 1 2.0 0.064
Dialysis buffer 3 500 6 RT 2 1.0 1.28 × 10– 4
Dialysis buffer 4 1000 6 4ºC 2 0.5 1.28 × 10– 7
Equilibration buffer 1 1000 6 4ºC 3 0 8.53× 10– 11
The final protein sample was concentrated with CentriconTM (30 kD MWCO) to a final volume of 0.85 ml and stored at –20 ºC. The refolding profile was analysed with SDS-PAGE and final protein concentration estimated by Bradford (Section 2.3.3).
2.6.3 Biotinylation of rabbit albumin 2.6.3.1 Materials
Reaction buffer [0.1 M NaH2PO4.2H2O, 0.15 M NaCl, 0.1% (w/v) NaN3, pH 7.5].
NaH2PO4.2H2O (7.8 g), NaCl (4.4 g) and NaN3 (0.5 g) were dissolved in 450 ml of dH2O, pH was adjusted to 7.5 and the volume was made up to 500 ml with dH2O.
NHS-biotin solution [10 mg/ml]. NHS-biotin (1 mg) was dissolved in DMSO (100 µl).
Rabbit albumin solution [20 mg/ml]. Rabbit albumin (10 mg) was dissolved in 500 ml of reaction buffer.
2.6.3.2 Method
The amount of NHS-biotin (MW 341.38) to be coupled to 10 mg of rabbit albumin at a ratio of 20:1 NHS-biotin is to rabbit albumin was derived to be 1 mg of NHS-biotin/10 mg rabbit albumin. The reaction was initiated by mixing NHS-biotin solution (100 µl) with rabbit
albumin solution (400 µl) and incubating on a rotary mixer (1h, RT). The mixture was loaded onto Sephadex G-25 size exclusion chromatography column pre-equilibrated with the reaction buffer (10 column volumes). The biotin-rabbit albumin complex was eluted with the reaction buffer by collecting fractions (1 ml). The A280 nm of the eluted fractions was determined for each fraction. The first set of fractions with A280 nm > 0.5 were pooled together (~ 4 ml total volume).
2.6.4 Biotinylation analysis of recombinant His6-PyBCCP and GST-PyBCCP 2.6.4.1 Materials
D-biotin solution [100 mM in 0.01 M NaOH]. D-biotin (0.24 g) was dissolved in 10 mM NaOH solution and sterilised by filtration through a 0.22 µm syringe filter. The solution was stored at –20ºC.
2×YT-D-biotin-kan-cam. 2×YT was prepared as described in Section 2.5.14.1. D-biotin, kanamycin and chloramphenicol were added to a final concentration of 0.2 mM, 34 µg/ml and 30 µg/ml respectively after autoclaving for Rosetta-pT-PyBCCP cells.
2×YT-D-biotin-amp. 2×YT was prepared as described in Section 2.5.14.1. D-biotin and ampicillin were added to a final concentration of 0.2 mM and 100 µg/ml respectively after autoclaving for BL21-pGX-PyBCCP cells and P. yoelii putative copper transporter gene fragment cloned into pGEX-4T-1 in BL21 E. coli strain.
2.6.4.2 Method
A similar expression protocol described in Section 2.6.1.2 was employed for this study except that the out-growth 2×YT media was used with or without 0.2 mM D-biotin. The overnight culture was grown to OD600 nm of approximately 0.6 in 2×YT ± 0.2 mM D-biotin containing the appropriate selective antibiotics (12 ml) and further split into two cultures (5 ml). One of the two was induced with IPTG (0.5 mM) and incubated (37ºC, 5 h or 18ºC, 16 h). For a negative control experiment, P. yoelii putative copper transporter gene fragment cloned into pGEX-4T-1 in BL21 E. coli strain was expressed using the same conditions as BL21-pGX- PyBCCP. Table 2.8 illustrates the conditions used.
Table 2.8 Culture treatment for biotinylation study
Culture set 1 2 3 4
0.5 mM IPTG – + – +
0.2 mM D-biotin – – + +
The cells were harvested as described in Section 2.6.1.2. The total cell lysate was resolved on 10% acrylamide gel by SDS-PAGE (Section 2.3.1) and electro-blotted onto nitrocellulose membrane (Section 2.3.2). The blot was blocked with fresh 5% (w/v) fat-free milk in TBS (Section 2.3.2.1), incubated with or without avidin-peroxidase or monoclonal anti-biotin IgG in the presence or absence of D-biotin (10 mM) or avidin (10 µg/ml) as negative control.
2.7 Bioinformatics methods