Dataset Information

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.

SUBMITTER: Lee SB

PROVIDER: S-EPMC4547976 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Production of bioactive chicken (Gallus gallus) follistatin-type proteins in E. coli.

Lee Sang Beum SB Park Sung Kwon SK Kim Yong Soo YS

AMB Express 20150825 1

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 ...[more]

PMID: 26302688

Similar Datasets

Project description:Despite decades of investigation, the function of interphotoreceptor retinoid binding protein (IRBP), the most abundant protein in the interphotoreceptor matrix of vertebrates, remains enigmatic. Roles for IRBP in the visual cycle of rod photoreceptors and in the independent visual cycle of cone photoreceptors have been suggested, yet very little is known of the biology of IRBP in cone-dominant retinas, such as those of diurnal birds. Our aim was to identify and characterize expression of the IRBP of the cone-dominant chicken (Gallus gallus domesticus).Chicken IRBP mRNA was identified by PCR cloning. Primary protein structure, genomic organization, and phylogenies were determined through comparative sequence analyses. Expression of IRBP mRNA was characterized by northern analysis and by in situ hybridization on cryosectioned chicken retina. Expression of the IRBP protein was characterized by western blotting and by indirect immunofluorescence on cryosectioned retina and on retinal whole mounts.The chicken IRBP gene encodes a secreted protein with a predicted 1,252 amino acid length. The gene structure for chicken IRBP resembles that of most other vertebrates, with four homologous, modular repeats and introns within only the fourth module. Each module is more homologous with the corresponding module in other species than it is with the remaining chicken modules. Chicken retinal tissue contains a single IRBP mRNA transcript of approximately 4.8 kb and western analysis of chicken retina shows a single major band of 140 kDa. Chicken IRBP mRNA is expressed exclusively by retinal photoreceptor cells and the intensity of the hybridization signal shows light/dark rhythmicity. The IRBP protein is localized to the interphotoreceptor matrix of the chicken retina and to intracellular regions of photoreceptors, with a spatial distribution indicating an association with cone outer segments.The high degree of conservation of IRBP's primary structure, genomic organization, and cell-specific expression within the retinas of all vertebrates examined to date, including those with cone-dominant retinas, implies a conserved role for IRBP in photoreceptor function and/or health. Expression of chicken IRBP and its mRNA are functionally regulated. This report provides a necessary first step to explore a specific function for IRBP in the cone visual cycle.

Dataset Information

Production of bioactive chicken (Gallus gallus) follistatin-type proteins in E. coli.

Publications

Production of bioactive chicken (Gallus gallus) follistatin-type proteins in E. coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets