High-level bacterial expression of a natively folded, soluble extracellular domain fusion protein of the human luteinizing hormone/chorionic gonadotropin receptor in the cytoplasm of Escherichia coli.
We have expressed the extracellular domain of the human luteinizing hormone/chorionic gonadotropin (hLH/CG) receptor as a fusion protein with thioredoxin in the cytoplasm of an Escherichia coli strain that contains mutations in both the thioredoxin reductase and glutathione reductase genes. The chimeric protein isolated following induction of expression is purified in a soluble form and binds hCG with an affinity approximating that of native receptor. This truncated form of the receptor displays the same specificity as intact hLH/CG receptor and does not bind human follicle stimulating hormone. This cytoplasmically produced protein is expressed at levels that exceed 10 mg/L. Expression of properly folded extracellular domain of the hLH/CG receptor in the cytoplasm of E. coli allows the facile and economic purification of large quantities of material. This will facilitate the determination of the structure of the hormone-binding domain of the glycoprotein receptor as well as the production of epitope-specific antibodies.
Lobel
L I
LI
Center for Reproductive Sciences and Department of Obstetrics and Gynecology, Columbia University, New York, NY 10032, USA.
Pollak
S
S
Klein
J
J
Lustbader
J W
JW
D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism; D001483:Base Sequence; D006063:Chorionic Gonadotropin / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000648:ultrastructure; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding; D011974:Receptors, LH / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D013879:Thioredoxins / Q000235:genetics
D000906:Antibodies; D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins