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

Lee SB, Park SK, Kim YS - AMB Express (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Nutrition, Food and Animal Sciences, University of Hawaii, 1955 East-West Rd., Honolulu, HI, 96822, USA, kusakn35@gmail.com.

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.

No MeSH data available.


Related in: MedlinePlus

Inhibition of MSTN, GDF11 or activin A activities by various FST-type proteins. HEK293 cells stably expressing (CAGA)12-luciferase gene construct were seeded on a 96-well culture, and grown for 24 h in DMEM with 10 % fetal calf serum, antibiotic and antimycotic. Medium was removed, and MSTN (1 nM), GDF11 (1 nM) or activin A (1 nM) plus various concentrations (180–0 nM) of FST-type proteins in DMEM were added to each well, followed by incubation for 24 h. Medium was removed, and luminescence substrate was added, followed by luminescence measurement. The error bars represent ± SEM (n = 6)
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Fig3: Inhibition of MSTN, GDF11 or activin A activities by various FST-type proteins. HEK293 cells stably expressing (CAGA)12-luciferase gene construct were seeded on a 96-well culture, and grown for 24 h in DMEM with 10 % fetal calf serum, antibiotic and antimycotic. Medium was removed, and MSTN (1 nM), GDF11 (1 nM) or activin A (1 nM) plus various concentrations (180–0 nM) of FST-type proteins in DMEM were added to each well, followed by incubation for 24 h. Medium was removed, and luminescence substrate was added, followed by luminescence measurement. The error bars represent ± SEM (n = 6)

Mentions: The abilities of MBP-FST288, MBP-NDFSD1/2, MBP-NDFSD1, and MBP-NDFSD1/1 to suppress MSTN, GDF11, and activin A were examined using pGL3-(CAGA)12 Luc-luciferase reporter assay (Fig. 3), and their potencies were compared each other, as well as to commercial recombinant human FST produced in eukaryotic cells (rhFST315/CHO, R&D Systems) or to MBP-FST315. It has been observed in our previous study that the presence of MBP as a fusion partner had no influence on the MSTN- or activin A-inhibitory capacity of FST315 (Lee et al. 2014). The removal of aggregates appeared in non-reduced condition of SDS-PAGE by gel-filtration did not significantly affect the MSTN- or activin A-inhibitory capacity of FST315 (Lee et al. 2014). Therefore, in measuring the bioactivities of FST-type proteins, we used amylose/heparin affinity-purified proteins without further purification.Fig. 3


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

Lee SB, Park SK, Kim YS - AMB Express (2015)

Inhibition of MSTN, GDF11 or activin A activities by various FST-type proteins. HEK293 cells stably expressing (CAGA)12-luciferase gene construct were seeded on a 96-well culture, and grown for 24 h in DMEM with 10 % fetal calf serum, antibiotic and antimycotic. Medium was removed, and MSTN (1 nM), GDF11 (1 nM) or activin A (1 nM) plus various concentrations (180–0 nM) of FST-type proteins in DMEM were added to each well, followed by incubation for 24 h. Medium was removed, and luminescence substrate was added, followed by luminescence measurement. The error bars represent ± SEM (n = 6)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4547976&req=5

Fig3: Inhibition of MSTN, GDF11 or activin A activities by various FST-type proteins. HEK293 cells stably expressing (CAGA)12-luciferase gene construct were seeded on a 96-well culture, and grown for 24 h in DMEM with 10 % fetal calf serum, antibiotic and antimycotic. Medium was removed, and MSTN (1 nM), GDF11 (1 nM) or activin A (1 nM) plus various concentrations (180–0 nM) of FST-type proteins in DMEM were added to each well, followed by incubation for 24 h. Medium was removed, and luminescence substrate was added, followed by luminescence measurement. The error bars represent ± SEM (n = 6)
Mentions: The abilities of MBP-FST288, MBP-NDFSD1/2, MBP-NDFSD1, and MBP-NDFSD1/1 to suppress MSTN, GDF11, and activin A were examined using pGL3-(CAGA)12 Luc-luciferase reporter assay (Fig. 3), and their potencies were compared each other, as well as to commercial recombinant human FST produced in eukaryotic cells (rhFST315/CHO, R&D Systems) or to MBP-FST315. It has been observed in our previous study that the presence of MBP as a fusion partner had no influence on the MSTN- or activin A-inhibitory capacity of FST315 (Lee et al. 2014). The removal of aggregates appeared in non-reduced condition of SDS-PAGE by gel-filtration did not significantly affect the MSTN- or activin A-inhibitory capacity of FST315 (Lee et al. 2014). Therefore, in measuring the bioactivities of FST-type proteins, we used amylose/heparin affinity-purified proteins without further purification.Fig. 3

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Nutrition, Food and Animal Sciences, University of Hawaii, 1955 East-West Rd., Honolulu, HI, 96822, USA, kusakn35@gmail.com.

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.

No MeSH data available.


Related in: MedlinePlus