Heterologous expression of soluble, active proteins in Escherichia coli: the human estrogen receptor hormone-binding domain as paradigm.
The human estrogen receptor ligand-binding domain (hER-E/F), including the distal F domain, has been expressed to high levels in a soluble, active form in Escherichia coli in order to facilitate biophysical studies. The ability of a series of vectors incorporating strong transcriptional and translational signals to provide an efficient expression system for hER-E/F was investigated. High-level expression was obtained from all of the vectors used in the study. Although the majority of hER-E/F protein was produced in insoluble form under standard bacterial culture conditions, hER-E/F could be produced in soluble, biologically active form by altering the sequence of the expressed protein and by varying the host culture conditions. Several parameters, including the presence of a His tag, growth temperature, and addition of ethanol and 17beta-estradiol to the growth medium were shown to have a positive effect on production of soluble hER-E/F. An optimized expression system capable of producing from 25 to 35 mg of biologically active hER-E/F in 1 liter of cell culture was designed, and a simple, rapid purification protocol for hER-E/F produced in this system was developed. Characterization of purified hER-E/F by Edman degradation and mass spectrometry verified the identity of the protein. The K(D) for 17beta-estradiol binding to purified hER-E/F was determined to be 0.6 +/- 0.1 nM. The parameters controlling soluble, heterologous protein production observed in this study may be generally applicable to the expression of other heterologous proteins in E. coli.
Nygaard
F B
FB
Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark.
Harlow
K W
KW
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D014158:Transcription, Genetic
D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D000431:Ethanol; D004958:Estradiol