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Production of Bioactive Rockfish (Sebastes schlegeli) Myostatin-1 Prodomain in an Escherichia coli System

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Abstract

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth in mammalian species, and its activity is inhibited by MSTN prodomain, the N-terminal part of proMSTN cleaved during post-translational MSTN processing. In fish, MSTN also appears to suppress fish muscle growth with its activity being inhibited by prodomain. The objective of this study was to produce bioactive MSTN-1 prodomain of rockfish (S. schlegeli), a commercial aquaculture species in East Asia, in E. coli using maltose binding protein (MBP) as a fusion partner. Rockfish MSTN-1 prodomain (sMSTN1pro) cDNA was cloned into the pMALc2x vector, and proteins (MBP-sMSTN1pro) were expressed in Rosetta-gami 2(DE3)pLysS cells by IPTG induction. The MBP-sMSTN1pro was expressed in soluble forms, and affinity purified using amylose resin. The affinity purified MBP-sMSTN1pro suppressed MSTN activity in vitro. The results suggest that MBP is probably a useful fusion partner in producing bioactive MSTN prodomains of various animal species in E. coli.

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Abbreviations

MSTN:

Myostatin

TGF-β:

Transforming Growth Factor-β

GASP1:

GDF-8-associated serum protein-1

FLRG:

Follistatin-related gene

hSGT:

human small glutamine-rich tetratricopeptide repeat-containing protein

MBP:

Maltose binding protein

PCR:

Polymerase chain reaction

sMSTN1pro:

sMSTN-1 prodomain

LB:

Luria-Bertani

NaCl:

Sodium chloride

IPTG:

Isopropyl-β-d-thiogalactoside

EDTA:

Ethylenediaminetetraacetic acid

SDS–PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TBST:

Tris-Buffered Saline Tween-20

IgG:

Immunoglobulin G

BCIP:

5-Bromo-4-chloro-3-indolyl phosphate

NBT:

Nitro blue tetrazolium

DMEM:

Dulbecco’s modified Eagle’s medium

GST:

Glutathione-s-transferase

Trx:

Thioredoxin

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Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0023056) and partly by USDA T-STAR grant (2004-34135-15196).

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Authors and Affiliations

  1. Division of Marine Molecular Biotechnology, Faculty of Marine Bioscience and Technology, Gangneung-Wonju National University, Gangneung, 210-702, Korea

    Sang Beum Lee, Mi-Jin Cho, Jeong Hwan Kim & Hyung-Joo Jin

  2. Department of Human Nutrition, Food and Animal Sciences, University of Hawaii, 1955 East–West Rd., Honolulu, HI, 96822, USA

    Yong Soo Kim

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  1. Sang Beum Lee
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  2. Mi-Jin Cho
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Correspondence to Hyung-Joo Jin.

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Lee, S.B., Cho, MJ., Kim, J.H. et al. Production of Bioactive Rockfish (Sebastes schlegeli) Myostatin-1 Prodomain in an Escherichia coli System. Protein J 30, 52–58 (2011). https://doi.org/10.1007/s10930-010-9301-1

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