Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15.
Oocyte-derived growth factors are critically involved in multiple ovarian processes via paracrine actions. Although recombinant proteins have been applied to dissect the physiological functions of these factors, variation of activities among different protein preparations remains an issue. To further elucidate the roles of one of these growth factors, bone morphogenetic protein 15 (BMP15), in mediating oocyte-regulated molecular and cellular events and to explore its potential clinical application, we engineered the human BMP15 sequence to efficiently produce bioactive recombinant human BMP15 (rhBMP15). The proteolytic cleavage site of the hBMP15 precursor was optimized to facilitate the production of the mature protein, and a FLAG-tag was placed at the N-terminus of the mature region to ease purification and avoid potential interference of the tag with the cystine knot structure. The rhBMP15 protein was purified using anti-FLAG M2 affinity gel. Our results demonstrated that the N-terminal tagged rhBMP15 was efficiently processed in HEK-293 cells. Furthermore, the purified rhBMP15 could activate SMAD1/5/8 and induce the transcription of genes encoding cumulus expansion-related transcripts (Ptx3, Has2, Tnfaip6 and Ptgs2), inhibitory SMADs (Smad6 and Smad7), BMP antagonists (Grem1 and Fst), activin/inhibin betaA (Inhba) and betaB (Inhbb) subunits, etc. Thus, our rhBMP15 containing a genetically modified cleavage sequence and an N-terminal FLAG-tag can be efficiently produced, processed and secreted in a mammalian expression system. The purified rhBMP15 is also biologically active and very stable, and can induce the expression of a variety of mouse granulosa cell genes.
Li
Qinglei
Q
Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Rajanahally
Saneal
S
Edson
Mark A
MA
Matzuk
Martin M
MM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D055430:Bone Morphogenetic Protein 15 / Q000235:genetics / Q000378:metabolism*; D002097:C-Reactive Protein / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000099153:Pentraxins
D055430:Bone Morphogenetic Protein 15; D002097:C-Reactive Protein; D009419:Nerve Tissue Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; D051785:Smad Proteins; D000099153:Pentraxins