Premium
Expression and characterization of two novel transporter orthologs, BOCT1 and BOCT2
Author(s) -
Bennett Katie M.,
Stoll James
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.813.4
Subject(s) - recombinant dna , affinity chromatography , monoclonal antibody , biochemistry , amino acid , fusion protein , chemistry , organic cation transport proteins , microbiology and biotechnology , hek 293 cells , transporter , biology , gene , antibody , genetics , enzyme
The objective of this study is to characterize two novel members of the organic cation transporter (OCT) family: BOCT1 and BOCT2. Primary aims include the expression of recombinant BOCT1 and BOCT2 protein in cells, functional characterization, and generation of monoclonal antibodies. The novel genes and two technical control transporters, OCT1 and OCTN2, were expressed as fusion proteins, stably transfected into HEK‐293 cells, and assayed for organic cation uptake. OCT1 caused a significant increase in choline (1.5‐fold), tetraethylammonium (2‐fold), and 1‐methyl–4‐phenylpyridinium (40‐fold) uptake, and OCTN2 caused a significant increase in carnitine (12‐fold) uptake. However, BOCT1 and BOCT2 did not show any effect, suggesting that they do not function in organic cation transport despite homology to the OCT family. In preparation for making monoclonal antibodies, a DNA fragment encoding ∼60 amino acids of both BOCT1 and BOCT2 was amplified via PCR and each cloned into the pRSET plasmid vector containing a 6‐His peptide tag sequence. Upon induction in E.coli, peptides accumulated in the insoluble inclusion body fractions, which were solubilized in 8M urea. The inclusion bodies were further purified by metal affinity chromatography, and the ∼12kD peptides were analyzed by SDS‐PAGE. Refolding conditions were optimized, and the peptides were prepared for immunization into mice. Funded by NIH R15 GM070578.