title: Over-expression in E. coli and purification of the human OCTN2 transport protein.
abstract: The OCTN2 cDNA amplified from human skin fibroblast was cloned in pET-41a(+) carrying the glutathione S-transferase (GST) gene. The construct pET-41a(+)-hOCTN2 was used to express the GST-hOCTN2 fusion protein in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained after 6 h of induction with IPTG at 28°C. The GST-hOCTN2 polypeptide was collected in the inclusion bodies and showed an apparent molecular mass on SDS-PAGE of 85 kDa. After solubilization with a buffer containing 0.8% sarkosyl and 3 M urea, the fusion protein was applied onto a Ni(2+)-chelating chromatography column. The purified GST-hOCTN2 was treated with thrombin, and the hOCTN2 was separated from the GST by size exclusion chromatography. After the whole procedure, a yield of about 0.2 mg purified protein per liter of cell culture was obtained. To improve the protein yield, hOCTN2 cDNA was subjected to codon bias. The second codon CGG was substituted with AAA; the substitution led to the mutation R2K in the hOCTN2 protein. hOCTN2(R2K) cDNA was cloned in pET-21a(+) carrying a C-terminal 6His tag. The resulting protein was expressed in E. coli Rosetta(DE3)pLysS and purified by Ni(2+)-chelating chromatography. A yield of about 3.5 mg purified protein per liter of cell culture was obtained with this procedure.
authors.affiliation: Department of Cell Biology, University of Calabria, Via P. Bucci 4c, 87036, Arcavacata di Rende, Italy. mgalluccio@unical.it
authors.forename: Michele
authors.initials: M
authors.lastname: Galluccio
authors.forename: Linda
authors.initials: L
authors.lastname: Amelio
authors.forename: Mariafrancesca
authors.initials: M
authors.lastname: Scalise
authors.forename: Lorena
authors.initials: L
authors.lastname: Pochini
authors.forename: Eckhard
authors.initials: E
authors.lastname: Boles
authors.forename: Cesare
authors.initials: C
authors.lastname: Indiveri
mesh_terms: D001709:Biotechnology / Q000379:methods; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D027701:Organic Cation Transport Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D000074058:Solute Carrier Family 22 Member 5
chemical_list: D003062:Codon; D027701:Organic Cation Transport Proteins; D011993:Recombinant Fusion Proteins; C492020:SLC22A5 protein, human; D000074058:Solute Carrier Family 22 Member 5; D005982:Glutathione Transferase