| Indiveri C, Iacobazzi V, Giangregorio N, Palmieri F.
(1998)
Biochemical and Biophysical Research Com,
249,
589-594 |
| Over expression & Renaturation |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| CO214(DE3) |
| 37.0 |
| 2 h |
| pMW172 |
| The plasmid was amplified in E. coli grown at 37􏰆C for 4 hours. Colonies containing the carnitine/acylcarnitine carrier coding sequence were identified by PCR. In order to ensure that no mutations had occurred in the sequence of the cloned carnitine/acylcarnitine carrier cDNA, the sequence was verified by the dideoxy chain termination method. Positive plasmids from these colonies were used to transform E. coli C0214.
tone-precipitated samples was performed in the presence of 0.1%
Bacterial expression. The over-production of the protein as inclu-sion bodies in the bacterial cytosol was accomplished as described before for the bovine 2-oxoglutarate carrier (16), except that the host cells were E. coli C0214(DE3), a mutant of E. coli C41(DE3) (see ref. 17). Control cultures containing the empty pMW172 vector were processed in parallel. After 2 h, unless otherwise specified, cells wereharvested by centrifugation and immediately used for the preparation of inclusion bodies as previously described (16). |
| Not Stated |
| OD n/a =
n/a |
| Not stated |
| None |
| Size-exclusion chromatography |
| insoluble |
| Column refolding: Size-exclusion chromatography |
| n/a |
| 800 ml of a buffer containing 2% (w/v) sarkosyl and 30 mM Pipes pH 7.0 |
| 100 mM NaCl/10 mM Pipes pH 7.0 |
| Size-exclusion chromatography |
| no tag |
| 7.0 |
| 0.0 |
| n/a |
| n/a |
| None |
| n/a |
| Purification of the recombinant carnitine/acylcarnitine carrier protein. Eight hundred microliters of the inclusion bodies in buffer A were mixed with 1 ml of a buffer containing 1% (w/v) Triton X-100, 0.5 mM MgCl2, 10 mM Pipes pH 7.0 and 15 U.I. DNAse. After 10 min incubation at 10􏰆C, the suspension was centrifuged at 30,000 g for 5 min; the pellet was solubilized in 800 ml of a buffer containing 2% (w/v) sarkosyl and 30 mM Pipes pH 7.0. After 10 min incubation at 0􏰆C, the suspension was centrifuged at 120,000 g for 30 min and the supernatant (extract) was collected. For the purification of the carnitine/acylcarnitine carrier, the inclusion body extract was mixed with 200 ml of 500 mM NaCl/10 mM Pipes pH 7.0 and applied on a Sephadex G-200 column (1.5 x 20 cm) preequilibrated with 100 mM
NaCl/10 mM Pipes pH 7.0; fractions of 4 ml were eluted with the same buffer. The 5th fraction was applied on a dry celite column (1 cm diameter containing 1 g dry resin). The column was washed with pressed 6 ml of 3% Triton X-100/10 mM Pipes pH 7.0 and the carnitine/ acylcarnitine carrier was then eluted by the same buffer supplemented with 4 mg/ml cardiolipin. A fraction of 2 ml was collected after the addition of the last buffer |
| Unspecified |
| None |
| None |
| n/a |
| 15 mg/l |
| n/a |
| n/a |