title: Binding studies using Pichia pastoris expressed human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins.
abstract: The aryl hydrocarbon receptor (AHR) is a transcription factor which activates gene transcription by binding to its corresponding enhancer as the heterodimer, which is consisted of AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT). Human AHR can be rather difficult to study, when compared among the AHR of other species, since it is relatively unstable and less sensitive to some ligands in vitro. Overexpression of human AHR has been limited to the baculovirus expression, which is costly and tedious due to the need of repetitive baculovirus production. Here we explored whether we could generate abundant amounts of human AHR and ARNT in a better overexpression system for functional study. We observed that human AHR and ARNT can be expressed in Pichia pastoris with yields that are comparable to the baculovirus system only if their cDNAs are optimized for Pichia expression. Fusion with a c-myc tag at their C-termini seems to increase the expression yield. These Pichia expressed proteins can effectively heterodimerize and form the ternary AHR/ARNT/enhancer complex in the presence of β-naphthoflavone or kynurenine. Limited proteolysis using thermolysin can be used to study the heterodimerization of these human AHR and ARNT proteins.
authors.affiliation: Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Yujuan
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Jinghang
authors.initials: J
authors.lastname: Xie
authors.affiliation: Department of Pharmacy, Qianfoshan Hospital of Shandong University, Jinan, Shandong, 250014, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Huang
authors.affiliation: Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Dong
authors.affiliation: Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Miki S
authors.initials: MS
authors.lastname: Park
authors.affiliation: Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA 95211, USA. Electronic address: wchan@pacific.edu.
authors.forename: William K
authors.initials: WK
authors.lastname: Chan
mesh_terms: D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003062:Codon; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding; D060066:Protein Interaction Maps; D055503:Protein Multimerization; D059748:Proteolysis; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D051792:Basic Helix-Loop-Helix Proteins; D003062:Codon; D018076:DNA, Complementary; D018336:Receptors, Aryl Hydrocarbon; D011994:Recombinant Proteins; D013820:Thermolysin; C111235:AHR protein, human; C497396:ARNT protein, human
keywords: Human AHR; Human ARNT; Pichia expression; Thermolysin