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.
Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark.
F B
FB
Nygaard
K W
KW
Harlow
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