| RRC ID |
2761
|
| 著者 |
Arai KY, Tsuchida K, Li C, Watanabe G, Sugino H, Taya K, Nishiyama T.
|
| タイトル |
Purification of recombinant activin A using the second follistatin domain of follistatin-related gene (FLRG).
|
| ジャーナル |
Protein Expr Purif
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| Abstract |
Activins are multifunctional growth factors belonging to the transforming growth factor-beta superfamily. Isolation of activins from natural sources requires many steps and only produces limited quantities. Even though recombinant preparations have been used in recent studies, purification of recombinant activins still requires multiple steps. To purify recombinant activin A, we have developed a simple method using the second follistatin domain of an activin-binding protein follistatin-related gene (FLRG). An affinity column was prepared with a partial FLRG fusion protein. The partial FLRG protein contained the second follistatin domain and the C-terminus acidic domain, and was tagged with six histidine residues at its N-terminus. The fusion protein was expressed in Escherichia coli and purified with nickel affinity column. Thereafter, the purified fusion protein was coupled to NHS-activated column. Recombinant activin A was produced in Chinese hamster ovary (CHO) cells, which were stably transfected with rat inhibin/activin betaA-subunit cDNA. After 48-h suspension culture of the cells in a serum free medium, the culture media was recovered and passed through the FLRG-coupled column. After washing with phosphate-buffered saline, bound protein was eluted out with an acidic buffer. Any significant contaminations were not detected when the purified protein was analyzed by SDS-PAGE. Apparent sizes of the protein were 14 and 28 kDa under the reduced and non-reduced conditions, respectively. Western blot analysis confirmed that the purified protein was activin A. The purified recombinant activin stimulated p3TP-lux reporter activity in CHO cells and follicle-stimulating hormone secretion from rat pituitary cells.
|
| 巻・号 |
49(1)
|
| ページ |
78-82
|
| 公開日 |
2006-9-1
|
| DOI |
10.1016/j.pep.2006.04.003
|
| PII |
S1046-5928(06)00112-4
|
| PMID |
16737827
|
| MeSH |
Activins / genetics
Activins / isolation & purification*
Activins / metabolism*
Animals
Cattle
Cells, Cultured
Cricetinae
Follistatin / genetics
Follistatin / metabolism*
Inhibin-beta Subunits / genetics
Inhibin-beta Subunits / isolation & purification*
Inhibin-beta Subunits / metabolism*
Inhibins / genetics
Inhibins / isolation & purification
Inhibins / metabolism
Protein Subunits / genetics
Protein Subunits / isolation & purification
Protein Subunits / metabolism
Rats
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / isolation & purification
Recombinant Fusion Proteins / metabolism
|
| IF |
1.513
|
| 引用数 |
5
|
|
WOS 分野
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
BIOCHEMICAL RESEARCH METHODS
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| リソース情報 |
| ラット |
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