Paper Information

Journal:   INTERNATIONAL JOURNAL OF FERTILITY AND STERILITY   SUMMER 2012 , Volume 6 , Number SUPPLEMENT 1; Page(s) 119 To 119.
 
Paper: 

CLONING AND EXPRESSION OF RECOMBINANT OVINE FSH HORMONE IN PICHIA PASTORIS

 
 
Author(s):  AHANJAN M.*, SANATI M.H., AMIRI YEKTA A., ESKANDARI M.P., FATEMI N., BAHRAMINEJAD E., EBRAHIMI S., AZIZZADEH PORMEHR L., GOURABI H.
 
* DEPARTMENT OF GENETICS, REPRODUCTIVE BIOMEDICINE RESEARCH CENTER, ROYAN INSTITUTE FOR REPRODUCTIVE BIOMEDICINE, ACECR, TEHRAN, IRAN
 
Abstract: 
Background: Follicle stimulating hormone is a heterodimeric protein composed of two subunits, a and b, which are linked noncovalently. The hypophysial gonadotropin FSH plays an important role in the regulation of oocyte maturation, and a key component for growth of ovulator follicles in ewes.
Materials and Methods: This study seeks to clone and express the ovine follicle stimulating hormone subunits in Pichia pastoris, which has numerous advantages over other systems for expression of eukaryotic proteins. The ORF region of alpha and beta fragments were amplified by PCR and inserted into PTZ57R/T vector and the follow subcloned in pPIC9 expression vector with a-factor signal peptide under the control of AOX1 promoter. The recombined plasmids were cotransformed into the P.
pastoris GS115 strain by electroporation. Incubation was continued for 102 hours at 29.5oC and 0.5% methanol was added every 24 hours during the period of incubation as an inducer. Finally, collected supernatant in during incubation were investigated by SDS-PAGE and Western blotting.
Results: Over results confirmed integration of linearized expression cassettes into the P. pastoris genome. Also expression of the recombinant protein was confirmed by SDS-PAGE and Western blotting.
Conclusion: Production of recombinant protein in eukaryotic expression systems such as P. pastoris is a reliable method for producing of therapeutic ovine FSH.
Whereas the secretion of the recombinant protein in supernatant could be cause the α-factor sequence as a signal peptide.
 
Keyword(s): RECOMBINANT FSH, CLONING, OVINE‚ PICHIA PASTORIS
 
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