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Expression and purification of the extracellular domain of the human follicle‐stimulating hormone receptor using E scherichia coli
Author(s) -
Zhang Xiaoyan,
Hong Shanshan,
Kang Yu,
Zheng Yufang,
Sun Hong,
Xu Congjian
Publication year - 2014
Publication title -
journal of obstetrics and gynaecology research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.597
H-Index - 50
eISSN - 1447-0756
pISSN - 1341-8076
DOI - 10.1111/jog.12203
Subject(s) - medicine , extracellular , escherichia coli , follicle , receptor , hormone , hormone receptor , follicle stimulating hormone receptor , endocrinology , microbiology and biotechnology , follicle stimulating hormone , biochemistry , biology , luteinizing hormone , gene , cancer , breast cancer
Aim 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 (h FSHR ). Methods Total RNA was isolated from normal human ovary tissue. c DNA for h FSHR ECD were amplified and subsequently ligated into the p ET32 a(+) vector. The plasmid vector construct was confirmed by polymerase chain reaction and sequencing. Expression in E scherichia coli R osetta ( DE3 ) p L ys S strain was induced by isopropyl‐thio‐β‐ D ‐thiogalactoside, and the recombinant products were purified by immuno‐affinity chromatography using an N i‐ NTA and H igh‐ Q column. The recombinant protein was confirmed by western blotting. Results Following induction, E . coli expressed a recombinant protein of approximately 65 k D a in size, whereas the non‐induced E . coli did not express the recombinant protein. The recombinant fragments purified using a H igh‐ Q column demonstrated a single band and an abundant yield. The recombinant protein was soluble and specifically recognized by an antibody for h FSHR . Additionally, four mutation sites were detected that resulted in amino acid shifts at position 112 A sn/ T hr, 197 G lu/ A la, 198 L eu/ V a l and 307 A la/ T hr. Conclusion The recombinant h FSHR 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.