High level expression of functional full length human thyroid hormone receptor beta 1 in insect cells using a recombinant baculovirus.
We have cloned the human thyroid hormone receptor beta 1 (hThR beta) from the human breast cancer cell line T47D using the PCR technique. A recombinant baculovirus transfer vector pVL1392/hThR beta was constructed and the full length receptor was expressed in the insect cell line Spodoptera frugiperda (Sf9). Approx. 10-15 x 10(6) receptors are expressed/cell which implies a production level of 2.5-4.0 mg hThR beta/l of cell culture. The expressed hThR beta displayed a single class of binding sites for T3 with high affinity. Western blot analysis using a polyclonal antibody indicated that the molecular weight of the baculovirus expressed receptor is approx. 50 kDa. Crude nuclear extract of hThR beta labeled with [125I]T3 sedimented as a 4 S peak on a glycerol gradient. No receptor could be detected in the cytoplasm indicating its proper translocation to the nuclear compartment. An oligonucleotide containing a palindromic thyroid hormone response element is specifically recognized and retarded in a gel-mobility-shift assay in the presence of nuclear extract of Sf9 cells expressing hThR beta. These data suggest that hThR beta expressed in Sf9 cells is functional and displays characteristics virtually indistinguishable from those of the thyroid hormone receptor (ThR) extracted from mammalian cells. Furthermore, the data indicate that the baculovirus expression system is adequate for large-scale production of receptor for detailed structural and functional studies.
Barkhem
T
T
Karo Bio AB, Huddinge, Sweden.
Carlsson
B
B
Simons
J
J
Möller
B
B
Berkenstam
A
A
Gustafsson
J A
JA
Nilsson
S
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D001943:Breast Neoplasms; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011988:Receptors, Thyroid Hormone / Q000235:genetics* / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D004247:DNA