title: Production of bioactive chicken (Gallus gallus) follistatin-type proteins in E. coli.
abstract: Follistatin (FST) is a cysteine-rich autocrine glycoprotein and plays an important role in mammalian prenatal and postnatal development. FST binds to and inhibit myostatin (MSTN), a potent negative regulator of skeletal muscle growth, and FST abundance enhances muscle growth in animals via inhibition of MSTN activity. The objective of this study was to produce biologically active, four chicken FST-type proteins in an Escherichia coli expression system. Gibson assembly cloning method was used to insert the DNA fragments of four FST-type proteins, designated as FST288, NDFSD1/2, NDFSD1, and NDFSD1/1, into pMALc5x vector downstream of the maltose-binding protein (MBP) gene, and the plasmids containing the inserts were eventually transformed into Shuffle E. coli strain for protein expression. We observed a soluble expression of the four MBP-fused FST-type proteins, and the proteins could be easily purified by the combination of amylose and heparin resin affinity chromatography. MBP-fused FST-type proteins demonstrated their affinity to anti-FST antibody. In an in vitro reporter gene assay to examine their potencies and selectivities to different ligands (MSTN, GDF11, and activin A), the four FST-type proteins (MBP-FST288, MBP-NDFSD1/2, MBP-NDFSD1, and MBP-NDFSD1/1) showed different potency and selectivity against the three ligands from each other. Ligand selectivity of each FST-type proteins was similar to its counterpart FST-type protein of eukaryotic origin. In conclusion, we could produce four FST-type proteins having different ligand selectivity in E. coli, and the results imply that economic production of a large amount of FST-type proteins with different ligand selectivity is possible to examine their potential use in meat-producing animals.
authors: Department of Human Nutrition, Food and Animal Sciences, University of Hawaii, 1955 East-West Rd., Honolulu, HI, 96822, USA, kusakn35@gmail.com.
authors: Sang Beum
authors: SB
authors: Lee
authors: Sung Kwon
authors: SK
authors: Park
authors: Yong Soo
authors: YS
authors: Kim