Expression, purification, renaturation and activation of fish myostatin expressed in Escherichia coli: facilitation of refolding and activity inhibition by myostatin prodomain.
Myostatin (growth and differentiation factor-8) is a member of the transforming growth factor-beta superfamily, is expressed mainly in skeletal muscle and acts as a negative growth regulator. Mature myostatin (C-terminal) is a homodimer that is cleaved post-translationally from the precursor myostatin, also yielding the N-terminal prodomain. We expressed in Escherichia coli three forms of fish myostatin: precursor, prodomain and mature. The three forms were over-expressed as inclusion bodies. Highly purified inclusion bodies were solubilized in a solution containing guanidine hydrochloride and the reducing agent DTT. Refolding (indicated by a dimer formation) of precursor myostatin, mature myostatin or a mixture of prodomain and mature myostatin was compared under identical refolding conditions, performed in a solution containing sodium chloride, arginine, a low concentration of guanidine hydrochloride and reduced and oxidized glutathione at 4 degrees C for 14 days. While precursor myostatin formed a reversible disulfide bond with no apparent precipitation, mature myostatin precipitated in the same refolding solution, unless CHAPS was included, and only a small proportion formed a disulfide bond. The trans presence of the prodomain in the refolding solution prevented precipitation of mature myostatin but did not promote formation of a dimer. Proteolytic cleavage of purified, refolded precursor myostatin with furin yielded a monomeric prodomain and a disulfide-linked, homodimeric mature myostatin, which remained as a latent complex. Activation of the latent complex was achieved by acidic or thermal treatments. These results demonstrate that the cis presence of the prodomain is essential for the proper refolding of fish myostatin and that the cleaved mature dimer exists as a latent form.
Funkenstein
Bruria
B
Department of Marine Biology and Biotechnology, Israel Oceanographic and Limnological Research, National Institute of Oceanography, P.O. Box 8030, Tel-Shikmona, Haifa 31080, Israel. bruria@ocean.org.il
Rebhan
Yanai
Y
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D029941:Fish Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D006358:Hot Temperature; D002479:Inclusion Bodies / Q000737:chemistry; D055435:Myostatin; D017510:Protein Folding; D020673:Protein Renaturation; D021541:Sea Bream / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification
D029941:Fish Proteins; D055435:Myostatin; D016212:Transforming Growth Factor beta