High level expression of a truncated chicken progesterone receptor in Escherichia coli.
Using a novel Escherichia coli system we have successfully overexpressed a region of the chicken progesterone receptor which encodes both the DNA- and hormone-binding domains. The expression system produces the truncated receptor fragment as an in-frame fusion with ubiquitin. This strategy greatly enhances both the solubility and stability of fusion proteins expressed in E. coli. Synthesis has been further improved by induction of the lambda PL promoter with nalidixic acid at low growth temperatures (less than or equal to 30 degrees C) rather than use of conventional heat induction protocols. We can produce 10 mg of receptor fragment/liter of cells using this system, and we estimate that at least 0.3 mg of this receptor material is biologically active, as assessed by DNA-binding and hormone-binding assays. Receptor produced in this manner is almost indistinguishable from authentic oviduct progesterone receptor using the criteria of hormone-binding specificity and affinity and binding to a progesterone response element. This expression system offers a cheap convenient method for the production of mg amounts of biologically active derivatives of progesterone receptor for biochemical studies.
Power
R F
RF
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Conneely
O M
OM
McDonnell
D P
DP
Clark
J H
JH
Butt
T R
TR
Schrader
W T
WT
O'Malley
B W
BW
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli; D010957:Plasmids; D011374:Progesterone / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D012995:Solubility; D014452:Ubiquitins
D004268:DNA-Binding Proteins; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D011374:Progesterone; D004247:DNA