| Jung ST, Kim MS, Seo JY, Kim HC, Kim Y.
(2003)
Protein Expression and Purification,
31,
240-246 |
| Purification & characterization |
| N-terminal GST and C-terminal hexahis |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 4 h |
| pET23c,pET25b,pET21a |
| All the purification and refolding procedures were carried out at 4 °C unless otherwise indicated. The transformants were grown in 1 L of LB medium containing 100 μg/ml of ampicillin at 37 °C until the OD600 of culture reached 0.6. For induction, 1 mM of isopropyl-1-thio-β- -galactopyranoside (IPTG) was added. After 4 h of induction, the transformants were harvested by centrifugation at 7000g for 20 min and resuspended in 60 ml of buffer A. Triton X-100 (1%; w/v) and DNase (0.1 mg/ml) were sequentially added to the lysates at intervals of 30 min during inverted mixing, and then sonication was repeated twice at 70% efficiency with a sonicator (Sonics & Materials). Inclusion body fractions were collected by centrifugation at 8000g for 20 min and washed in 60 ml of buffer B. The washed inclusion bodies were solubilized in 60 ml of buffer C, incubated overnight with constant mixing, and filtered through a 0.45 μm syringe filter (Sartorius). The solubilized inclusion bodies were purified using the Ni–NTA agarose resins (Qiagen) according to the manufacturer’s recommendations. The purity of the LOX and LOXL proteins was analyzed by SDS–PAGE. |
| IPTG |
| OD 0.6 =
600 |
| Sonication |
| Detergents |
| Ni-NTA agrose chromatography |
| insoluble |
| Dialysis |
| 2 M urea, and 10 mM K2HPO4, pH 8.2. |
| 8 M Urea, 10 mM K2HPO4, pH 8.2, and 3 mM β-mercaptoethanol |
| 10 mM K2HPO4, pH 9.6, 200 μM CuCl2, and 2% sodium N-lauroylsarcosinate and buffer 10 mM K2HPO4, pH 9.6, and 5 μM CuCl2 and 10 mM K2HPO4, pH 7.0 |
| Ni-NTA agrose chromatography |
| no |
| 9.6 |
| 4.0 |
| n/a |
| overnight |
| None |
| n/a |
| In an attempt to refold the proteins denatured by urea, the protein samples were subjected to consecutive dialyses. First, the purified proteins were diluted to 100 μg/ml in buffer D. The diluted proteins were dialyzed overnight first in buffer E containing 2% of sodium N-lauroylsarcosinate and then against buffer F. The proteins were further dialyzed twice against buffer D overnight. The concentration of dialyzed protein samples was determined by the BCA method [21]. After dialysis, the protein solutions were aliquoted and lyophilized using a Freeze dryer (Labconco). Trehalose was added to the protein solutions at a final concentration of 10 mM before lyophilization. Samples were frozen at −40 °C, dried for 24 h, capped within vacuum, and then stored at −20 °C.
Residual chemicals in the purified proteins, such as sodium N-lauroylsarcosinate, imidazol, β-mercaptoethanol, and urea, were analyzed at 210 nm by HPLC equipped with a μ-Bondapack C18 reverse phase column (Waters Associates; 300 × 3.9 mm). The column was pre-equilibrated with the buffer of 0.2 mM K2HPO4, pH 7.0, and the samples were eluted in the solvent of 100% acetonitrile with a linear gradient of 0–40% over 20 min at a flow rate of 1 ml/min. |
| Amine Oxidise |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |