Refolding and purification of unprocessed porcine myostatin expressed in Escherichia coli.
Myostatin is a member of the transforming growth factor-beta (TGF-beta) superfamily, and it acts as a negative regulator for skeletal muscle growth. Like many other TGF-beta family member proteins, the mature form of myostatin is a homodimer that is processed post-translationally from a precursor form of myostatin. Since the presence of a prodomain is essential for proper folding and homodimer assembly for some members of the TGF-beta superfamily, we compared the refolding in vitro of porcine unprocessed and mature myostatin over-expressed in Escherichia coli as inclusion bodies. A high alkaline buffer solution containing a mild anionic detergent and a reducing agent was used to solubilize the myostatin inclusion bodies. An optimal condition for refolding was obtained by rapid dilution of the solubilized protein in a buffer system containing reduced and oxidized glutathione, and subsequent incubation at 4 degrees C for at least 7 days. The unprocessed porcine myostatin demonstrated reversible disulfide bond formation after refolding, a characteristic of the native form of myostatin. In contrast, the mature myostatin formed aggregates that did not demonstrate reversible disulfide bond formation in the refolding condition used in this study. These results demonstrate the importance of the myostatin prodomain in facilitating the proper folding of mature myostatin. Reaction of the refolded, unprocessed myostatin with furin, an endopeptidase cleaving between paired basic residues, yielded prodomain and mature myostatin, demonstrating that the unprocessed myostatin is a substrate for furin. The prodomain did not form disulfide bond formation but the mature myostatin demonstrated reversible disulfide-linked homodimer formation. It is concluded that myostatin prodomain facilitates the proper folding of myostatin, and the refolded, native form of unprocessed myostatin could be obtained in high yield (15%) after E. coli expression as inclusion bodies.
Jin
Hyung-Joo
HJ
Department of Human Nutrition, Food and Animal Sciences, University of Hawaii, 1955 East-West Rd., Honolulu, HI 96822, USA.
Dunn
Michael A
MA
Borthakur
Dulal
D
Kim
Yong Soo
YS
D000818:Animals; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D045683:Furin / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D018482:Muscle, Skeletal / Q000502:physiology; D055435:Myostatin; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D013552:Swine; D016212:Transforming Growth Factor beta / Q000737:chemistry* / Q000235:genetics
C525911:MSTN protein, human; D055435:Myostatin; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D045683:Furin