Bacterial Expression of a Natively Folded Extracellular Domain Fusion Protein of the hFSH Receptor in the Cytoplasm of Escherichia coli

We have expressed the extracellular domain of the hFSH receptor as a fusion protein with thioredoxin in the cytoplasm of an Escherichia coli strain that contains mutations in both the thioredoxin reductase and the glutathione reductase genes. The chimeric protein isolated following induction of expr...

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Bibliographic Details
Published in:Protein expression and purification 2002-06, Vol.25 (1), p.124-133
Main Authors: Lobel, Leslie, Pollak, Susan, Lustbader, Brandie, Klein, Jeffrey, Lustbader, Joyce W.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
bacterial expression
Base Sequence
Blotting, Western
Cytoplasm - metabolism
Electrophoresis, Polyacrylamide Gel
Epitopes
Escherichia coli - metabolism
follicle-stimulating hormone
fusion protein
hFSH receptor
Humans
Models, Genetic
Molecular Sequence Data
Protein Binding
Protein Folding
Protein Structure, Tertiary
Receptors, FSH - chemistry
Recombinant Fusion Proteins - metabolism
Thioredoxins - metabolism
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Summary:We have expressed the extracellular domain of the hFSH receptor as a fusion protein with thioredoxin in the cytoplasm of an Escherichia coli strain that contains mutations in both the thioredoxin reductase and the glutathione reductase genes. The chimeric protein isolated following induction of expression was purified in a soluble form and binds hFSH with an affinity approximating that of native receptor. This truncated form of the receptor displays the same specificity as intact receptor and does not bind hCG. The protein is expressed at levels that exceed 5 mg/L in the bacterial cytoplasm. Expression of the properly folded extracellular domain of the hFSH receptor in the cytoplasm of E. coli allows the facile and economical purification of large quantities of material. This will facilitate the determination of the structure of the hormone-binding domain of this glycoprotein receptor as well as the production of epitope-specific antibodies.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.2002.1618