Cloning And Prokaryotic Expression Of The Bovine Follistatin CDNA Gene

Follistatin, a kind of secreted protein, is known to antagonise the function of several members of the TGF-beta (transforming growth factor-beta) superfamily including Myostatin, the most powerful inhibitor of muscle growth characterised to date. Follistatin antagonises Myostatin by a direct protein-protein interaction, which prevents Myostatin from executing its inhibitory effect on muscle development. Follistatin inhibits myostatin's ability to bind to receptors and has applications for promoting muscle growth by a combined function of muscle fiber hyperplasia (the number of muscle fibers) and hypertrophy (the thickness of fibers).To integrate my work to other researches in our laboratory on myostatin gene, the purpose of my research is cloning and prokaryotic expression of the bovine Follistatin cDNA gene.Bovine Follistatin cDNA gene was cloned by RT-PCR using total RNA as template, which was extracted from bovine ovary by Trizol Total RNA Extract Kit. The purified bovine Follistatin cDNA was inserted into pET-30a vector to construct the prokaryotic expression vector pET-30a-fs2. Sequence analysis data indicated that 99% homology was indentified between the published bovine Follistatin cDNA gene and my two-directional sequenced products. The sequence data also shows that the open reading frame of the pET-30a-fs2 is correct. The recombinant expression plasmid was transformed into BL21(DE3) and gene expression was induced by administration of IPTG. Expression was detected by SDS-PAGE and confirmed by Western blot analysis, which shows that the yield of the target protein was approximately 30% of the total bacterial protein.Follistatin may have pharmacological applications for treating human muscle diseases as well as applications as an agent for enhancing muscle growth of livestock.