Over-expression in E. coli and purification of the human OCTN1 transport protein.
The hOCTN1 amplified from skin fibroblast RNA was cloned in pET-28a(+) or in pH6EX3 plasmid. The encoded recombinant hOCTN1 resulted in a 6-His tagged fusion protein with a 34 or 21 amino acid extra N-terminal sequence in the pET-28a(+)-hOCTN1 or in the pH6EX3-hOCTN1 constructs, respectively. Both constructs were used to express the hOCTN1 in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained with the pH6EX3-hOCTN1 after 6h of induction with IPTG at 28 degrees C. The expressed protein with an apparent molecular mass of 54kDa, was collected in the insoluble fraction of the cell lysate. Further improvement was obtained using the E. coli RosettaGami2(DE3)pLysS strain to express the protein encoded by pH6EX3-hOCTN1. After 6h of induction with IPTG at 28 degrees C, hOCTN1 accounted for 30% of the total protein in the insoluble pellet. This protein fraction was washed with Triton X-100 and deoxycholate, solubilized with a buffer containing 0.8% Sarkosyl, 3M urea and applied to a Ni2+-chelating chromatography column. The homogeneously purified hOCTN1 was eluted with a buffer containing 50 mM imidazole, 0.1% Triton X-100 and 50 mM 2-mercaptoethanol. A yield of about 3mg purified protein per liter of cell culture was obtained.
Galluccio
Michele
M
Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036 Arcavacata di Rende (CS), Italy.
Pochini
Lorena
L
Amelio
Linda
L
Accardi
Rosita
R
Tommasino
Massimo
M
Indiveri
Cesare
C
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008623:Mercaptoethanol / Q000737:chemistry; D008954:Models, Biological; D008969:Molecular Sequence Data; D027701:Organic Cation Transport Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D027981:Symporters
D027701:Organic Cation Transport Proteins; D011993:Recombinant Fusion Proteins; C109807:SLC22A4 protein, human; D027981:Symporters; D008623:Mercaptoethanol