248 PRODUCTION OF FLAGGED RECOMBINANT BOVINE BMP15 TO IMPROVE BOVINE IN VITRO EMBRYO PRODUCTION SYSTEMS

Abstract

Current in vitro systems for bovine embryo production are inefficient and produce embryos with lower viability than their in vivo-derived counterparts. Recent reports demonstrate that in vitro bovine oocyte maturation systems could benefit from the addition of oocyte-secreted factors, specifically GDF9 and BMP15 (Gilchrist et al. 2007 Theriogenology 67, 6–15). The long-term goal of this work is to produce species-specific recombinant oocyte-secreted factors capable of improving bovine embryo production in vitro. In the current project, the objective was to produce a cell line that expresses recombinant bovine BMP15. This protein is first translated as a large precursor peptide consisting of propeptide and mature regions, which are enzymatically cleaved to form the active mature protein. The wild-type BMP15 gene was cloned using reverse transcriptase PCR with RNA obtained from bovine ovarian tissue. For improved detection and purification of the active form of the recombinant protein, a detectable FLAG tag sequence (DYKDDDDK) was incorporated into the wild-type BMP15 gene by PCR and cloned into pCDNA expression vector. The FLAG tag was introduced immediately 3′ of the cleavage site at the N-terminal portion of the mature protein to produce recombinant FLAG-tagged BMP15 (rbFL-BMP15). To ensure efficient production of the mature protein, a Kozak sequence was inserted 5′ of the start ATG and the cleavage site altered to be recognised by PACE/furin enzymes, which are endogenously expressed in most mammalian cells including HEK-293 cells (Li et al. 2009 Mol. Hum. Reprod. 15, 779–788). Following sequencing to verify transcript fidelity, pCDNA-rbFL-BMP15 was transfected into HEK-293 cells, and mature protein production was detected by Western blot analysis. Cells plated at 85% confluency were transfected with Lipofectamine 2000, and lysates were harvested 48 h post-transfection. The presence of bovine rbFL-BMP-15 in cell lysates was confirmed by Western blot using the anti-FLAG antibody. Ongoing experiments will test the bioactivity of the purified rbFL-BMP15 by evaluating activation of the SMAD 1/5 pathway via Western blot for phosphorylated SMAD 1/5. After a biologically active protein is confirmed, purified protein will be collected for testing during in vitro maturation of bovine oocytes. We anticipate the species-specific form of oocyte-secreted factors will further enhance in vitro embryo production systems beyond that reported using heterologous factors.