P-205: Production of Recombinant Fish FSH Hormone in Pichia Pastoris
Authors
Abstract:
Background: Follicle-stimulating hormone (FSH) belongs to the family of glycoprotein hormones that composing alpha and beta subunits with non-covalently bonds. This hormone involve in regulation of the reproductive processes such as gamete generation and follicular growth. Injection of the hormone in most of fish species increases 17 beta-estradiol production by ovarian tissue and also stimulates 11-keto testosterone production in testes. The purpose of this research is production of recombinant fish FSH hormone (rfFSH) by Pichia pastoris expression system under post-translational modification. Materials and Methods: The alpha and beta fish FSH chains was separately cloned in pTZ57R / T vector and was subcloned into pHILS1 expression vector. This vector (pHILS1) was transformed into competent cell (Pichia pastoris yeast) by electroporation. Transformed clones were suspended in BMMY liquid medium and induced by 0.5% methanol to high levels of expression. The medium was centrifuged at 14000 rpm for 4 minutes for separating Yeast cell from media. Finally, the separated media containing target protein concentrated by Amicon Ultra centrifugal filter for detecting protein by SDS-PAGE and Western blotting techniques. Results: The integration of both vector including alpha and beta FSH genes in the yeast genome was confirmed by PCR with pHILS1 vector-specific primers (5'AOXI and 3'AOXI). Also expression and secretion of rfFSH hormone was confirmed by SDS-PAGE and Western blotting techniques. Conclusion: Pichia pastoris can be an ideal protein expression system for producing a protein with high specific biological activity and correct folding pattern, also it can be used as a model for increasing expression level of glycoprotein hormones by recombinant DNA technology.
similar resources
P-191: Cloning and Expression of Recombinant Ovine FSH Hormone in Pichia Pastoris
Background: Follicle stimulating hormone is a heterodimeric protein composed of two subunits, α and ß, 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 sub...
full textEffect of Codon Optimisation on the Production of Recombinant Fish Growth Hormone in Pichia pastoris
This study was established to test the hypothesis of whether the codon optimization of fish growth hormone gene (FGH) based on P. pastoris preferred codon will improve the quantity of secreted rFGH in culture supernatant that can directly be used as fish feed supplements. The optimized FGH coding sequence (oFGH) and native sequence (nFGH) of giant grouper fish (Epinephelus lanceolatus) were clo...
full textEvaluation of Sorbitol-Methanol Co-Feeding Strategy on Production of Recombinant Human Growth Hormone in Pichia Pastoris
Recombinant protein production in Pichia pastoris is based on alcohol oxidase promoterswhich are regulated by methanol. However, the use of methanol has several disadvantages,which is why current trends in bioprocess development with Pichia pastoris (P. pastoris) arefocusing on methanol mixed feeding strategies. This work aimed to develop a new experimentalmethod and compare the effect of vario...
full textEvaluation of Sorbitol-Methanol Co-Feeding Strategy on Production of Recombinant Human Growth Hormone in Pichia Pastoris
Recombinant protein production in Pichia pastoris is based on alcohol oxidase promoterswhich are regulated by methanol. However, the use of methanol has several disadvantages,which is why current trends in bioprocess development with Pichia pastoris (P. pastoris) arefocusing on methanol mixed feeding strategies. This work aimed to develop a new experimentalmethod and compare the effect of vario...
full textTargeted Deletion of Los1 Homologue Affects the Production of a Recombinant Model Protein in Pichia pastoris
Background: The methylotrophic yeast Pichia pastoris is an appealing production host for a variety of recombinant, including biologics. In this sense, various genetic- and non-genetic-based techniques have been implemented to improve the production efficiency of this expression platform. Los1 (loss of supression) encodes a non-essential nuclear tRNA exporter in Saccharomyces cerevisiae, which i...
full textProduction of recombinant human epidermal growth factor in Pichia pastoris
This study was carried out to express human epidermal growth factor (hEGF) in Pichia pastoris GS115. For this aim, the hEGF gene was cloned into the pPIC9K expression vector, and then integrated into P. pastoris by electroporation. ELISA-based assay showed that the amount of hEGF secreted into the medium can be affected by the fermentation conditions especially by culture medium, pH and tempera...
full textMy Resources
Journal title
volume 7 issue 3
pages 117- 117
publication date 2013-09-01
By following a journal you will be notified via email when a new issue of this journal is published.
Hosted on Doprax cloud platform doprax.com
copyright © 2015-2023