Texas A&M University
Burns, Gregory Willis.
Production and Functional Analysis of Recombinant Bovine Morphogenic Protein 15.
Degree: MS, Biomedical Sciences, 2013, Texas A&M University
After 40 years of research, in vitro systems for mammalian embryo production produce lower quality embryos than those derived from in vivo sources. Recent reports have demonstrated that in vitro bovine oocyte maturation systems benefit from the addition of oocyte secreted factors, specifically Bone Morphogenic Protein 15 (BMP15) from heterologous sources. However, known amino acid sequence variation and species-specific patterns of post-translational glycosylation lead us to hypothesize that utilization of bovine-specific oocyte secreted factors would be more beneficial than the observed effects of heterologous factors.
To test this hypothesis, wild type, bovine BMP15 was cloned using reverse transcriptase PCR from RNA obtained from bovine ovarian tissue. For improved detection and purification of the biologically active recombinant protein, a FLAG tag peptide sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) was incorporated into the wild type BMP15 gene by PCR. This modified protein was cloned into the pCDNA 3 mammalian expression vector. HEK-293 (human embryonic kidney 293) and FBK (fetal bovine kidney) cell lines were transfected via electroporation and then selected to homogeneity.
Collection and purification of rbFL-BMP15 from conditioned medium was accomplished by incubation with anti-FLAG affinity gel and the use of 3X FLAG peptide for elution. Peptides of 15.4 kDa and 17 kDa were noted from the human HEK-293 transfected cell line, while in contrast, bovine FBK cells produced a single 17 kDa protein. Bioactivity and BMP receptor signaling specificity were ascertained using in vitro treatment of HeLa cells and Western blotting for the BMP signaling molecule phosphorylated-SMAD 1/5. Inhibition of this signaling cascade using dorsomorphin, a selective bone morphogenic protein receptor I inhibitor, demonstrated the purified proteins served as BMP15-like agonists.
To examine the impact of our purified, bovine-specific peptides on oocyte maturation, cumulus oocyte complexes were in vitro matured for 24 hours in the presence of 100 ng/ml recombinant human BMP15 or rbFL-BMP15 from human or bovine cell lines. Real time quantitative PCR analysis of BMP15 stimulated genes, PTGS2 and TSG6, revealed statistically significant increases in transcript level for treatment with human BMP15 by a Dunnett’s test (p<0.05). In this report, however, we failed to detect a significant affect of rbFL-BMP15 on the gene expression of in vitro mature cumulus oocyte complexes at 24 hours with 100 ng/ml rbFL-BMP15. Future studies including differing time points and concentrations, along with the addition of GDF9 to form a possible heterodimer should be investigated for the possibility of improving bovine oocyte in vitro maturation.
Advisors/Committee Members: Long, Charles R (advisor), Golding, Michael C (committee member), Li, Qinglei (committee member).
Subjects/Keywords: BMP15; IVM; TGFB; recombinant protein; FLAG; BMPRI; ART; cattle; bovine; COC; oocyte
to Zotero / EndNote / Reference
APA (6th Edition):
Burns, G. W. (2013). Production and Functional Analysis of Recombinant Bovine Morphogenic Protein 15. (Masters Thesis). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/151804
Chicago Manual of Style (16th Edition):
Burns, Gregory Willis. “Production and Functional Analysis of Recombinant Bovine Morphogenic Protein 15.” 2013. Masters Thesis, Texas A&M University. Accessed January 16, 2021.
MLA Handbook (7th Edition):
Burns, Gregory Willis. “Production and Functional Analysis of Recombinant Bovine Morphogenic Protein 15.” 2013. Web. 16 Jan 2021.
Burns GW. Production and Functional Analysis of Recombinant Bovine Morphogenic Protein 15. [Internet] [Masters thesis]. Texas A&M University; 2013. [cited 2021 Jan 16].
Available from: http://hdl.handle.net/1969.1/151804.
Council of Science Editors:
Burns GW. Production and Functional Analysis of Recombinant Bovine Morphogenic Protein 15. [Masters Thesis]. Texas A&M University; 2013. Available from: http://hdl.handle.net/1969.1/151804