The bacterial expression vector encoding codon-optimized his-tagged human apoA1 has been previously described [17 (link)]. All point mutations and deletions were created using the QuickChange II Mutagenesis Kit (Thermo Fisher). All mutations were confirmed by DNA sequencing. Expression plasmids were transformed into E. coli BL21 dE3 pLysS and protein expression was induced in shaking cultures by overnight incubation with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside at room temperature. The resulting cellular pellet was resuspended in B-PER lysis solution (Thermo Fisher) containing Lysozyme, DNaseI, and a protease inhibitor cocktail. The cellular debris was removed by centrifugation and the supernatant was diluted into PBS containing 3 M guanidine-HCl. The denatured histidine-tagged apoA1 was purified using Ni Sepharose HP resin (Amersham Biosciences) followed by imidazole elution. Fractions containing recombinant apoA1 were extensively dialyzed against PBS and analyzed for purity by SDS-PAGE and Coomassie Blue staining. Only samples with >95% purity were used. When indicated apoA1 was reduced in 10 mM DTT, and reductive methylation was performed with 100 mM N-ethylmaleimide (NEM) at 37°C for 1 hr under nitrogen gas, followed by dialysis in PBS.
B per lysis solution
Manufactured by Thermo Fisher Scientific
B-PER lysis solution is a proprietary buffer designed for the extraction of recombinant proteins from E. coli cells. It is a ready-to-use solution that effectively disrupts bacterial cell walls and membranes to release soluble proteins for further purification or analysis.
Automatically generated - may contain errors
3 protocols using b per lysis solution
1
Purification of Recombinant Histidine-tagged ApoA1
2
Dot-Blot Analysis of S. aureus Lysates
3
Purification of Recombinant Bacterial Glycosidase
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The wild-type BgaD-D
enzyme and mutant proteins were heterologously produced and purified.
Briefly, the plasmids containing the wild-type and mutant genes were
transferred into E. coli BL21 (DE3) competent cells.
After growth on LB agar plates (containing 100 μg/mL ampicillin),
colonies were inoculated for overnight cultivation. Then 1% overnight
culture was inoculated into fresh LB medium (containing 100 μg/mL
ampicillin) and incubated at 37 °C. When the cell density reached
about 0.6 at 600 nm, expression of the recombinant proteins was induced
with 1 mM isopropyl-β-d -thiogalactopyranoside.
Subsequently, the cells were cultured overnight at 30 °C and
harvested by centrifugation. Cell pellets were washed with 20 mM Tris-HCl
buffer (pH 8.0) and lysed with B-PER lysis solution (Thermo Scientific)
for 1 h at room temperature. The cell debris was removed by centrifugation.
The supernatant was mixed with HIS-Select Nickel Affinity Gel and
incubated at 4 °C overnight. Unbound proteins were washed away
with 20 mM Tris-HCl (pH 8.0), 50 mM NaCl (buffer A); the recombinant
proteins were eluted with buffer A containing 100 mM imidazole. Then
imidazole was removed by ultracentrifugation with a cutoff of 30 kDa
(Merck).
enzyme and mutant proteins were heterologously produced and purified.
Briefly, the plasmids containing the wild-type and mutant genes were
transferred into E. coli BL21 (DE3) competent cells.
After growth on LB agar plates (containing 100 μg/mL ampicillin),
colonies were inoculated for overnight cultivation. Then 1% overnight
culture was inoculated into fresh LB medium (containing 100 μg/mL
ampicillin) and incubated at 37 °C. When the cell density reached
about 0.6 at 600 nm, expression of the recombinant proteins was induced
with 1 mM isopropyl-β-
Subsequently, the cells were cultured overnight at 30 °C and
harvested by centrifugation. Cell pellets were washed with 20 mM Tris-HCl
buffer (pH 8.0) and lysed with B-PER lysis solution (Thermo Scientific)
for 1 h at room temperature. The cell debris was removed by centrifugation.
The supernatant was mixed with HIS-Select Nickel Affinity Gel and
incubated at 4 °C overnight. Unbound proteins were washed away
with 20 mM Tris-HCl (pH 8.0), 50 mM NaCl (buffer A); the recombinant
proteins were eluted with buffer A containing 100 mM imidazole. Then
imidazole was removed by ultracentrifugation with a cutoff of 30 kDa
(Merck).
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