PMID- 7929368
OWN - NLM
STAT- MEDLINE
DCOM- 19941122
LR  - 20210212
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 269
IP  - 42
DP  - 1994 Oct 21
TI  - Baculovirus expression of the Ah receptor and Ah receptor nuclear translocater. 
      Evidence for additional dioxin responsive element-binding species and factors 
      required for signaling.
PG  - 26464-71
AB  - In an effort to facilitate the structural and biochemical analyses of the Ah 
      receptor (AHR) and the Ah receptor nuclear translocator (ARNT), a baculovirus 
      system was developed to express microgram-milligram quantities of the human 
      version of these proteins. To simplify purification, a polyhistidine tag was 
      cloned at their C termini so that the recombinant proteins could be specifically 
      adsorbed to nickel-nitriloacetic acid-Sepharose. Expression studies revealed that 
      approximately 23% of the overexpressed AHR was recovered in cell extracts with 
      the remaining 77% forming insoluble aggregates. ARNT was found to be more 
      soluble, with 90% recovery from cell extracts and only 10% aggregation. 
      Photoaffinity labeling and gel shift assays demonstrated that the recombinant 
      proteins bound ligand, heterodimerized, and recognized their cognate "dioxin 
      response element" (DRE) in a manner similar to their native counterparts. 
      Coexpression of the AHR and ARNT in Sf9 cells resulted in the in vivo generation 
      of heterodimers that bound the DRE in the absence of ligand. Studies with the 
      nickel-nitriloacetic acid-purified recombinant proteins demonstrated that the AHR 
      and ARNT could bind DRE only when reconstituted with a heat-sensitive factor(s) 
      present in soluble extracts from a variety of cell types. Use of these proteins 
      also demonstrated the existence of at least three AHR-dependent DRE-binding 
      species, suggesting that the AHR can bind to DRE in at least three distinct 
      conformations.
FAU - Chan, W K
AU  - Chan WK
AD  - Department of Molecular Pharmacology and Biological Chemistry, Northwestern 
      University Medical School, Chicago, Illinois 60611.
FAU - Chu, R
AU  - Chu R
FAU - Jain, S
AU  - Jain S
FAU - Reddy, J K
AU  - Reddy JK
FAU - Bradfield, C A
AU  - Bradfield CA
LA  - eng
GR  - ES-05703/ES/NIEHS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (ARNT protein, human)
RN  - 0 (Affinity Labels)
RN  - 0 (DNA-Binding Proteins)
RN  - 0 (Dioxins)
RN  - 0 (Proteins)
RN  - 0 (Receptors, Aryl Hydrocarbon)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Transcription Factors)
RN  - 138391-32-9 (Aryl Hydrocarbon Receptor Nuclear Translocator)
SB  - IM
MH  - Affinity Labels
MH  - Animals
MH  - Aryl Hydrocarbon Receptor Nuclear Translocator
MH  - Baculoviridae/genetics
MH  - Base Sequence
MH  - DNA-Binding Proteins/*metabolism
MH  - Dioxins/*pharmacology
MH  - Humans
MH  - Molecular Sequence Data
MH  - *Protein Biosynthesis
MH  - Proteins/analysis
MH  - Receptors, Aryl Hydrocarbon/analysis/*biosynthesis/chemistry
MH  - Recombinant Proteins/*biosynthesis
MH  - Spodoptera
MH  - *Transcription Factors
EDAT- 1994/10/21 00:00
MHDA- 1994/10/21 00:01
CRDT- 1994/10/21 00:00
PHST- 1994/10/21 00:00 [pubmed]
PHST- 1994/10/21 00:01 [medline]
PHST- 1994/10/21 00:00 [entrez]
AID - S0021-9258(18)47217-1 [pii]
PST - ppublish
SO  - J Biol Chem. 1994 Oct 21;269(42):26464-71.