Purification and characterization of biologically active recombinant human Eppin expressed in Escherichia coli.
BACKGROUND
Eppin (epididymis protease inhibitor) appears to play an important role in primate fertility. However, the function of Eppin and its antibody in men and its relationship with men's infertility are poorly studied. To reveal the significance and possibility of detection of anti-Eppin antibody in clinical infertilty cases, we developed an Escherichia coli expression system for the expression of biologically active human Eppin.


METHODS
The human Eppin gene was cloned into PET-28a( )+ vector after induction with 0.5 mmol/L isopropy-beta-D-thiogalactoside (IPTG) at 26 degrees C for 4 hours, and the expressed fusion protein His6-Eppin was purified by Ni2+ affinity chromatography. Afterwards, six female 8-week-old Balb/c mice were immunized with purified His6-Eppin for three weeks. Their sera were collected and polyclonal antibodies against His6-Eppin were purified, all of which were further verified by Western-blot and immunofluorescence analysis.


RESULTS
About 18.33 mg His6-Eppin was obtained from 1-L flask culture. The produced polyclonal antibodies against His6-Eppin recognized the Eppin protein both in human epididymis and in HEK293T cells by over-expression of the recombinant human Eppin.


CONCLUSIONS
The purified His6-Eppin protein has biological activity, which might be a candidate for clinical diagnosis of infertility and development of male immuno-contraceptive agents.
Zhu
Qing-yi
QY
Department of Urology, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, China.
Gu
Xiao-jian
XJ
Yang
Jin
J
Wang
Jun-hong
JH
Tang
Bo
B
Wu
Hong-fei
HF
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D053491:Proteinase Inhibitory Proteins, Secretory / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification*
C431553:Eppin protein, human; D053491:Proteinase Inhibitory Proteins, Secretory; D011993:Recombinant Fusion Proteins