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Purification and Reconstitution of Recombinant Myostatin Latent Complex
Author(s) -
Zhang Hong,
Trill John,
Johnson Kathleen,
Scott Gilbert,
Kull Frederick,
Johanson Kyung
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.535.3
Subject(s) - myostatin , furin , recombinant dna , cleavage (geology) , chemistry , chinese hamster ovary cell , biochemistry , protein precursor , biology , microbiology and biotechnology , receptor , gene , enzyme , paleontology , fracture (geology)
Myostatin (GDF‐8) is a member of the TGF b super family. It is expressed in skeletal muscle and acts as a negative regulator of skeletal muscle growth. Myostatin is synthesized as pre‐pro‐myostatin and secreted as an inactive pro‐myostatin, where pro‐domain is processed from mature myostatin by a furin‐like protease. In vivo, myostatin is in circulation as an inactive latent complex (LC) which consists of pro‐domain and mature myostatin. It was shown that active mature myostatin was released from inactive latent form upon cleavage of pro‐domain by BMP‐1. To study the proteolytic processing of LC, full length myostatin was expressed as a fusion protein with an N‐terminal 6His/GB1tag with Tev cleavage site in a CHO secretion system. Myostatin was captured using Ni‐NTA chromatography. However, it was a mixture of various forms, 1) incomplete furin cleavage, 2) intact or cleaved pro‐domain at BMP‐1 cleavage site or C‐terminus truncated pro‐domain, and 3) intact or C‐terminus truncated mature domain. Every component was tightly aggregated together. To obtain an intact recombinant latent complex (rLC), the mixture was treated with furin and Tev, adjusted to 6M Gdn‐HCl to dissociate the aggregation, then passed through Ni‐NTA to both remove the His/GB1 tag and to release the tag‐cleaved pro‐domain. Using C8 RP‐HPLC, mature myostatin was isolated from the mixture. When the pro‐domain and mature myostatin were mixed in 6M Gdn‐HCl, dialyzed, then loaded onto a sizing column in PBS, only the intact pro‐domain was complexed with mature myostatin forming an intact rLC. This rLC was used as a substrate for proteolytic study.