Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: The mouse carnitine transporter OCTN3

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• 作者：Mariafrancesca Scalise¹ ; Michele Galluccio¹ ; Lorena Pochini ; Cesare Indiveri ; ^{indiveri@unical.it}
• 关键词：TEA ; tetraethylammonium ; PLP ; pyridoxal 5-phosphate ; MTSET ; [2-(trimethylammonium)ethyl]methanethiosulfonate ; p-OHMB ; p-hydroxymercuribenzoate ; GABA ; ¦Ã-aminobutyric acid ; ANTP ; adenosine 5&prime ; -(¦Â ; ¦Ã-imido)triphosphate
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2012
• 出版时间：25 May, 2012
• 年：2012
• 卷：422
• 期：1
• 页码：59-63
• 全文大小：480 K

文摘

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 Ni²⁺-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 [³H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K_m for carnitine was 36 ¦ÌM. [³H]carnitine transport was inhibited by carnitine analogues and some Cys and NH₂ 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.