E. coli SHuffle T7 Express strain (New England Biolabs), which is an enhanced BL21 derivative engineered to express a chromosomal copy of the disulfide bond isomerase DsbC (Chen et al. 1999 (link)) in the cytoplasm constitutively, was transformed with the rKAA-1 expression vector pET28a-rKAA1 by a conventional method. A transformant was cultured with 1 L of LB media at 37 °C until OD600 reached 0.5, and then IPTG was added at a final concentration of 0.1 mM. After continuously cultured at 20 °C for 16 h, the bacteria cells were harvested by centrifugation at 10,000 g for 20 min. After fracturing the cell by sonication, the His-tagged rKAA-1 (His-rKAA-1) expressed into soluble fraction was purified by His GraviTrap (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instruction. After purification, His-rKAA-1 was dialyzed thoroughly against ultrapure water to remove imidazole which was contained in the elution buffer. Twenty-five milligrams of His-rKAA-1 was then applied to thrombin treatment using a Thrombin Cleavage Capture Kit (Merck) to disconnect the tag peptide from the recombinant according to the manufacturer’s instruction. After removal of thrombin by the above kit, the digested fraction was applied to His GraviTrap to exclude the cleaved His-tag peptide and undigested His-rKAA-1. Generated rKAA-1 was then dialyzed thoroughly against ultrapure water.
Shuffle t7 express strain
Manufactured by New England Biolabs
The SHuffle T7 Express strain is a bacterial expression strain designed for the production of disulfide bond-containing proteins. It features the SHuffle cytoplasmic disulfide bond isomerase, which facilitates the formation of disulfide bonds in the cytoplasm.
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2 protocols using shuffle t7 express strain
1
Purification of Recombinant Protein rKAA-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
E. coli SHuffle T7 Express strain (New England Biolabs), which is an enhanced BL21 derivative engineered to express a chromosomal copy of the disulfide bond isomerase DsbC (Chen et al. 1999 (link)) in the cytoplasm constitutively, was transformed with the rKAA-1 expression vector pET28a-rKAA1 by a conventional method. A transformant was cultured with 1 L of LB media at 37 °C until OD600 reached 0.5, and then IPTG was added at a final concentration of 0.1 mM. After continuously cultured at 20 °C for 16 h, the bacteria cells were harvested by centrifugation at 10,000 g for 20 min. After fracturing the cell by sonication, the His-tagged rKAA-1 (His-rKAA-1) expressed into soluble fraction was purified by His GraviTrap (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instruction. After purification, His-rKAA-1 was dialyzed thoroughly against ultrapure water to remove imidazole which was contained in the elution buffer. Twenty-five milligrams of His-rKAA-1 was then applied to thrombin treatment using a Thrombin Cleavage Capture Kit (Merck) to disconnect the tag peptide from the recombinant according to the manufacturer’s instruction. After removal of thrombin by the above kit, the digested fraction was applied to His GraviTrap to exclude the cleaved His-tag peptide and undigested His-rKAA-1. Generated rKAA-1 was then dialyzed thoroughly against ultrapure water.
+ Expand
E. coli SHuffle T7 Express strain (New England Biolabs), which is an enhanced BL21 derivative engineered to express a chromosomal copy of the disulfide bond isomerase DsbC (Chen et al. 1999 (link)) in the cytoplasm constitutively, was transformed with the rKAA-1 expression vector pET28a-rKAA1 by a conventional method. A transformant was cultured with 1 L of LB media at 37 °C until OD600 reached 0.5, and then IPTG was added at a final concentration of 0.1 mM. After continuously cultured at 20 °C for 16 h, the bacteria cells were harvested by centrifugation at 10,000 g for 20 min. After fracturing the cell by sonication, the His-tagged rKAA-1 (His-rKAA-1) expressed into soluble fraction was purified by His GraviTrap (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instruction. After purification, His-rKAA-1 was dialyzed thoroughly against ultrapure water to remove imidazole which was contained in the elution buffer. Twenty-five milligrams of His-rKAA-1 was then applied to thrombin treatment using a Thrombin Cleavage Capture Kit (Merck) to disconnect the tag peptide from the recombinant according to the manufacturer’s instruction. After removal of thrombin by the above kit, the digested fraction was applied to His GraviTrap to exclude the cleaved His-tag peptide and undigested His-rKAA-1. Generated rKAA-1 was then dialyzed thoroughly against ultrapure water.
2
Purification of Recombinant Protein rKAA-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
E. coli SHuffle T7 Express strain (New England Biolabs), which is an enhanced BL21 derivative engineered to express a chromosomal copy of the disulfide bond isomerase DsbC (Chen et al. 1999 (link)) in the cytoplasm constitutively, was transformed with the rKAA-1 expression vector pET28a-rKAA1 by a conventional method. A transformant was cultured with 1 L of LB media at 37 °C until OD600 reached 0.5, and then, IPTG was added at a final concentration of 0.1 mM. After continuously cultured at 20 °C for 16 h, the bacteria cells were harvested by centrifugation at 10,000g for 20 min. After fracturing the cell by sonication, the His-tagged rKAA-1 (His-rKAA-1) expressed into soluble fraction was purified by His GraviTrap (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instruction. After purification, His-rKAA-1 was dialyzed thoroughly against ultrapure water to remove imidazole which was contained in the elution buffer. Twenty-five milligrams of His-rKAA-1 was then applied to thrombin treatment using a Thrombin Cleavage Capture Kit (Merck) to disconnect the tag peptide from the recombinant according to the manufacturer’s instruction. After removal of thrombin by the above kit, the digested fraction was applied to His GraviTrap to exclude the cleaved His-tag peptide and undigested His-rKAA-1. Generated rKAA-1 was then dialyzed thoroughly against ultrapure water.
+ Expand
E. coli SHuffle T7 Express strain (New England Biolabs), which is an enhanced BL21 derivative engineered to express a chromosomal copy of the disulfide bond isomerase DsbC (Chen et al. 1999 (link)) in the cytoplasm constitutively, was transformed with the rKAA-1 expression vector pET28a-rKAA1 by a conventional method. A transformant was cultured with 1 L of LB media at 37 °C until OD600 reached 0.5, and then, IPTG was added at a final concentration of 0.1 mM. After continuously cultured at 20 °C for 16 h, the bacteria cells were harvested by centrifugation at 10,000g for 20 min. After fracturing the cell by sonication, the His-tagged rKAA-1 (His-rKAA-1) expressed into soluble fraction was purified by His GraviTrap (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instruction. After purification, His-rKAA-1 was dialyzed thoroughly against ultrapure water to remove imidazole which was contained in the elution buffer. Twenty-five milligrams of His-rKAA-1 was then applied to thrombin treatment using a Thrombin Cleavage Capture Kit (Merck) to disconnect the tag peptide from the recombinant according to the manufacturer’s instruction. After removal of thrombin by the above kit, the digested fraction was applied to His GraviTrap to exclude the cleaved His-tag peptide and undigested His-rKAA-1. Generated rKAA-1 was then dialyzed thoroughly against ultrapure water.
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