Production of chicken follistatin 315 and chicken follistatin 315 fused to a chicken IgY Fc fragment in Escherichia coli

Abstract

Follistatin (Fst) is an autocrine glycoprotein that binds to multiple members of the transforming growth factor family to regulate various physiological processes. Fst has shown to bind myostatin, a potent negative regulator of skeletal muscle growth. Inhibition of myostatin activity by Fst treatment enhanced muscle growth, as well as ameliorates symptoms of muscular dystrophy in animal models, illustrating the potential of Fst as a therapeutic agent to improve muscle growth in animal agriculture or to treat muscle wasting conditions or disease in humans. Therefore, we designed a study to produce biologically active recombinant chicken FST315 (MBP-chFST315) and a chFST315-Chicken IgY constant domain (3-4) fusion protein (MBP-chFST315-Fc(3-4)) in an Escherichia coli host. Complex folding patterns along with multiple disulfide bonds of Fst had made it a difficult challenge to express bioactive Fst protein in E. coli. In this study, we compared combination of vector systems and E. coli strains in order to produce a soluble form of chFST315. Disulfide bond formation of chFST315 is necessary for correct protein folding. Therefore, we expressed the chFST315 protein in either a system that utilizes a periplasmic expression strategy or a genetically modified E. coli system that is capable of disulfide bond formation in the cytoplasm. Periplasmic expression of MBP-chFST315 using the pMAL-p5x vector system failed to produce a soluble recombinant protein. However, when a cytoplasmic expression vector pMAL-c5x was combined with SHuffle® E. coli strain, we were successful in producing a soluble form of MBP-chFST315 and MBP-chFST315-Fc(3-4). The MBP-chFST315 was able to bind to myostatin and to a lesser extent to activin in an in vitro binding assay. MBP-chFST315 inhibited myostatin activity in an in vitro gene reporter assay. The recombinant MBP-chFST315 will be useful in investigating the potential of Fst in improving skeletal muscle growth of chicken as well as in future studies of investigating signaling pathways involved in skeletal muscle hypertrophy.