【资源】一个pQE30系统表达蛋白纯化案例
丁香园论坛
2610
偶然发现的一个pQE30系统表达蛋白纯化案例，帖出来和大家分享。
Purification of LF.
诱导与表达
E. coli SG13009(pREP4) carrying the recombinant plasmid pPG-LF1 was grown at 37°C in Luria broth with 100 μg of ampicillin and 25 μg of kanamycin per ml at 250 rpm.
When the A600 reached 1.0, isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 1 mM.
After 5 h of induction, the cells were harvested by centrifugation at 4,000 × g for 20 min.
样品处理
For the purification of protein from 2 liters of culture, the pellet was resuspended in 50 ml of sonication buffer (50 mM Na phosphate [pH 7.8], 300 mM NaCl). Lysozyme (1 mg/ml) was added to the slurry and incubated on ice for 30 min. Phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM.
Cells were sonicated at 4°C (1-min bursts, 1 min of cooling, 200 to 300 W) for five cycles. The lysate was centrifuged at 10,000 × g for 30 min. The supernatant was mixed with 8 ml of a 50% Ni-nitriloacetic acid resin previously equilibrated with sonication buffer.
亲和层析
The slurry was packed into a column (5.0 by 1.6 cm) and allowed to settle. The matrix was washed first with sonication buffer followed by wash buffer (50 mM Na phosphate [pH 6.0], 500 mM NaCl, 10% glycerol).
The column was washed until the A280 of the flowthrough was less than 0.01 (approximately 50 ml). Protein was eluted with a linear gradient of 15 ml each of 0 and 500 mM imidazole chloride in elution buffer (50 mM Na phosphate [pH 7.0], 100 mM NaCl, 10% glycerol).
Fractions of 1 ml were collected and analyzed on an SDS-10% PAGE gel. rLF eluted at a gradient of 100 to 250 mM imidazole chloride. Affinity-purified protein possessed full-length rLF, and approximately 40% degraded LF as determined by SDS-PAGE and Western blotting with anti-LF antibodies (Fig. 2).
样品浓缩和分子筛层析
The fractions containing LF were pooled and dialyzed against T10E5 buffer (10 mM Tris and 5 mM ETDA [pH 8.0]) overnight. The dialyzed sample was concentrated by Centricon-30 (Amicon) to a volume of 1 ml.
Concentrated sample was loaded onto a Sephacryl S-200 (Pharmacia) gel filtration column (100 by 1.6 cm) previously equilibrated with T10E5 buffer. Void volume was allowed to pass through, and 1-ml fractions were then collected.
The rLF eluted immediately after the void volume, while the mobility of the degraded products was retarded. This resulted in approximately 90% pure rLF with a very small amount of degraded LF.
离子交换层析
The fractions containing LF were pooled and loaded onto the Resource-Q (Pharmacia) anion-exchange column previously equilibrated with T10E5 buffer. Protein was eluted with a linear gradient of 0 to 1 M NaCl in T10E5 buffer (15 ml each). Fractions of 1 ml each were collected. The protein eluted at a gradient of 250 to 300 mM NaCl.
The purified LF was dialyzed against 10 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer (pH 7.0) containing 50 mM NaCl and was frozen at −70°C in aliquots. The fold purification of LF at different column stages was determined by calculating the amount of protein required to kill 50% of J774A.1 cells (50% effective concentration [EC50]) when they were incubated with PA (1 μg/ml) at 37°C (Table 1).
The protein was measured by the method described by Lowry et al.. By using this procedure, rLF was purified to homogeneity with 3,054-fold purification compared to the cytosolic preparation.
Purification of LF.
诱导与表达
E. coli SG13009(pREP4) carrying the recombinant plasmid pPG-LF1 was grown at 37°C in Luria broth with 100 μg of ampicillin and 25 μg of kanamycin per ml at 250 rpm.
When the A600 reached 1.0, isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 1 mM.
After 5 h of induction, the cells were harvested by centrifugation at 4,000 × g for 20 min.
样品处理
For the purification of protein from 2 liters of culture, the pellet was resuspended in 50 ml of sonication buffer (50 mM Na phosphate [pH 7.8], 300 mM NaCl). Lysozyme (1 mg/ml) was added to the slurry and incubated on ice for 30 min. Phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM.
Cells were sonicated at 4°C (1-min bursts, 1 min of cooling, 200 to 300 W) for five cycles. The lysate was centrifuged at 10,000 × g for 30 min. The supernatant was mixed with 8 ml of a 50% Ni-nitriloacetic acid resin previously equilibrated with sonication buffer.
亲和层析
The slurry was packed into a column (5.0 by 1.6 cm) and allowed to settle. The matrix was washed first with sonication buffer followed by wash buffer (50 mM Na phosphate [pH 6.0], 500 mM NaCl, 10% glycerol).
The column was washed until the A280 of the flowthrough was less than 0.01 (approximately 50 ml). Protein was eluted with a linear gradient of 15 ml each of 0 and 500 mM imidazole chloride in elution buffer (50 mM Na phosphate [pH 7.0], 100 mM NaCl, 10% glycerol).
Fractions of 1 ml were collected and analyzed on an SDS-10% PAGE gel. rLF eluted at a gradient of 100 to 250 mM imidazole chloride. Affinity-purified protein possessed full-length rLF, and approximately 40% degraded LF as determined by SDS-PAGE and Western blotting with anti-LF antibodies (Fig. 2).
样品浓缩和分子筛层析
The fractions containing LF were pooled and dialyzed against T10E5 buffer (10 mM Tris and 5 mM ETDA [pH 8.0]) overnight. The dialyzed sample was concentrated by Centricon-30 (Amicon) to a volume of 1 ml.
Concentrated sample was loaded onto a Sephacryl S-200 (Pharmacia) gel filtration column (100 by 1.6 cm) previously equilibrated with T10E5 buffer. Void volume was allowed to pass through, and 1-ml fractions were then collected.
The rLF eluted immediately after the void volume, while the mobility of the degraded products was retarded. This resulted in approximately 90% pure rLF with a very small amount of degraded LF.
离子交换层析
The fractions containing LF were pooled and loaded onto the Resource-Q (Pharmacia) anion-exchange column previously equilibrated with T10E5 buffer. Protein was eluted with a linear gradient of 0 to 1 M NaCl in T10E5 buffer (15 ml each). Fractions of 1 ml each were collected. The protein eluted at a gradient of 250 to 300 mM NaCl.
The purified LF was dialyzed against 10 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer (pH 7.0) containing 50 mM NaCl and was frozen at −70°C in aliquots. The fold purification of LF at different column stages was determined by calculating the amount of protein required to kill 50% of J774A.1 cells (50% effective concentration [EC50]) when they were incubated with PA (1 μg/ml) at 37°C (Table 1).
The protein was measured by the method described by Lowry et al.. By using this procedure, rLF was purified to homogeneity with 3,054-fold purification compared to the cytosolic preparation.
