Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: The mouse carnitine transporter OCTN3
- Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende (Italy)
Highlights: Black-Right-Pointing-Pointer mOCTN3 transport protein has been cloned in pET-21a(+) and over-expressed in Escherichia coli. Black-Right-Pointing-Pointer The expressed mOCTN3 has been purified to homogeneity by Ni-chelating chromatography. Black-Right-Pointing-Pointer The protein solubilised in Triton X-100 has been reconstituted in liposomes. Black-Right-Pointing-Pointer Recombinant mOCTN3 catalyses transport of carnitine by a uniport mode. -- Abstract: pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni{sup 2+}-chelating chromatography with a yield of 2 mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [{sup 3}H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K{sub m} for carnitine was 36 {mu}M. [{sup 3}H]carnitine transport was inhibited by carnitine analogues and some Cys and NH{sub 2} reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.
- OSTI ID:
- 22207864
- Journal Information:
- Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 1 Vol. 422; ISSN 0006-291X; ISSN BBRCA9
- Country of Publication:
- United States
- Language:
- English
Similar Records
Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis
Muscle contraction increases carnitine uptake via translocation of OCTN2