High level expression of biologically active estrogen receptor in Saccharomyces cerevisiae.
Biochemical over-expression of the human estrogen receptor was achieved using a Saccharomyces cerevisiae expression system. The receptor was produced as a novel ubiquitin fusion protein. This fusion protein is short lived in the cell and is processed to produce unfused receptor shortly after folding. Conventional high copy expression plasmids produced receptor to about 0.04% of the total soluble protein. By incorporating a defective leu2 allele into these vectors, an additional 5-fold increase in receptor production was obtained. The recombinant receptor was undergraded, soluble and biologically active. Conventional methods of disrupting cells using glass beads had a detrimental effect on the ability of the receptor to bind hormone. Enzymatic digestion of the cell wall followed by hypotonic shock liberates the receptor that quantitatively binds estrogen.
McDonnell
D P
DP
Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.
Nawaz
Z
Z
Densmore
C
C
Weigel
N L
NL
Pham
T A
TA
Clark
J H
JH
O'Malley
B W
BW
D000483:Alleles; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004967:Estrogens / Q000378:metabolism; D015870:Gene Expression; D005800:Genes, Fungal; D006801:Humans; D010957:Plasmids; D011233:Precipitin Tests; D011960:Receptors, Estrogen / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D004967:Estrogens; D011960:Receptors, Estrogen; D001616:beta-Galactosidase