title: Production of bioactive chicken follistatin315 in Escherichia coli.
abstract: Follistatin (FST) binds to myostatin (MSTN), a potent negative regulator of skeletal muscle growth. Inhibition of MSTN activity by FST treatment has shown to enhance muscle growth as well as ameliorate symptoms of muscular dystrophy in animal models, illustrating the potential of FST as an agent to enhance 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 (chFST315) in an Escherichia coli host. Since FST contains multiple intramolecular disulfide bonds, we expressed chFST315 protein in either a system that utilizes a periplasmic expression strategy, or a genetically modified E. coli system (SHuffle strain) that is capable of disulfide bond formation in the cytoplasm. Periplasmic expression of chFST315 using the pMAL-p5x vector system, which was designed to express maltose-binding protein (MBP) fusion protein, failed to produce a soluble recombinant protein. However, cytoplasmic expression of chFST315 using pMAL-c5x vector in SHuffle E. coli strain resulted in a soluble expression of the recombinant protein (MBP-chFST315). Combination of heparin and amylose resin affinity chromatography yielded about 6 mg/L purified MBP-chFST315. The purified MBP-chFST315 showed binding affinity to MSTN and activin in a pull-down assay, as well as inhibited MSTN and activin activity in an in vitro reporter gene assay. In conclusion, results of the study demonstrate that for the first time a recombinant, biologically active FST molecule can be produced in a soluble form in E. coli. The ability to produce FST in a cost-effective system is expected to allow us to investigate the potentials of FST as an agent to improve skeletal muscle growth of meat producing animals via suppression of MSTN.
authors.affiliation: Department of Human Nutrition, Food and Animal Sciences, University of Hawaii, 1955 East-west Rd., Honolulu, HI, 96822, USA.
authors.forename: Sang Beum
authors.initials: SB
authors.lastname: Lee
authors.forename: Rocky
authors.initials: R
authors.lastname: Choi
authors.forename: Sung Kwon
authors.initials: SK
authors.lastname: Park
authors.forename: Yong Soo
authors.initials: YS
authors.lastname: Kim
mesh_terms: D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D038681:Follistatin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D055435:Myostatin / Q000037:antagonists & inhibitors / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012995:Solubility
chemical_list: D038681:Follistatin; D055435:Myostatin; D011994:Recombinant Proteins