title: Expression and purification of the extracellular domain of the human follicle-stimulating hormone receptor using Escherichia coli.
abstract: OBJECTIVE
  Although much is known about the structure and biological functions of follicle-stimulating hormone (FSH) receptor (FSHR), the interaction of FSHR and FSH has been challenging to characterize due to the limited quantity of active FSHR protein produced by simple methods. The goal of this study was to express and purify the extracellular domain (ECD) of human FSHR (hFSHR).
  
  
  METHODS
  Total RNA was isolated from normal human ovary tissue. cDNA for hFSHR ECD were amplified and subsequently ligated into the pET32a(+) vector. The plasmid vector construct was confirmed by polymerase chain reaction and sequencing. Expression in Escherichia coli Rosetta (DE3) pLysS strain was induced by isopropyl-thio-β-D-thiogalactoside, and the recombinant products were purified by immuno-affinity chromatography using an Ni-NTA and High-Q column. The recombinant protein was confirmed by western blotting.
  
  
  RESULTS
  Following induction, E. coli expressed a recombinant protein of approximately 65 kDa in size, whereas the non-induced E. coli did not express the recombinant protein. The recombinant fragments purified using a High-Q column demonstrated a single band and an abundant yield. The recombinant protein was soluble and specifically recognized by an antibody for hFSHR. Additionally, four mutation sites were detected that resulted in amino acid shifts at position 112 Asn/Thr, 197 Glu/Ala, 198 Leu/Val and 307 Ala/Thr.
  
  
  CONCLUSIONS
  The recombinant hFSHR ECD protein was expressed and purified. This method could be easily scaled for increased production and may facilitate additional applications utilizing FSHR in assisted reproductive technology, a contraceptive FSH vaccine and FSHR-targeted therapeutic agents used to treat ovarian cancer.
authors.affiliation: Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China; Department of Obstetrics and Gynecology of Shanghai Medical College, Fudan University, Shanghai, China; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Shanghai, China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Zhang
authors.forename: Shanshan
authors.initials: S
authors.lastname: Hong
authors.forename: Yu
authors.initials: Y
authors.lastname: Kang
authors.forename: Yufang
authors.initials: Y
authors.lastname: Zheng
authors.forename: Hong
authors.initials: H
authors.lastname: Sun
authors.forename: Congjian
authors.initials: C
authors.lastname: Xu
mesh_terms: D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D020641:Polymorphism, Single Nucleotide; D011962:Receptors, FSH / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins
chemical_list: D011962:Receptors, FSH; D011994:Recombinant Proteins
keywords: bacterial expression; follicle-stimulating hormone; follicle-stimulating hormone receptor; purification; single nucleotide polymorphism