Purification of recombinant activin A using the second follistatin domain of follistatin-related gene (FLRG)

Koji Y. Arai, Kunihiro Tsuchida, Chun Mei Li, Gen Watanabe, Hiromu Sugino, Kazuyoshi Taya, Toshio Nishiyama

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Activins are multifunctional growth factors belonging to the transforming growth factor-β 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 βA-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.

Original languageEnglish
Pages (from-to)78-82
Number of pages5
JournalProtein Expression and Purification
Volume49
Issue number1
DOIs
Publication statusPublished - 01-01-2006

Fingerprint

Follistatin
Activins
Genes
Proteins
Cricetulus
Follistatin-Related Proteins
Ovary
Inhibins
activin A
Gene Fusion
Serum-Free Culture Media
Follicle Stimulating Hormone
Transforming Growth Factors
Nickel
Histidine
Culture Media
Polyacrylamide Gel Electrophoresis
Intercellular Signaling Peptides and Proteins
Suspensions
Buffers

All Science Journal Classification (ASJC) codes

  • Biotechnology

Cite this

Arai, Koji Y. ; Tsuchida, Kunihiro ; Li, Chun Mei ; Watanabe, Gen ; Sugino, Hiromu ; Taya, Kazuyoshi ; Nishiyama, Toshio. / Purification of recombinant activin A using the second follistatin domain of follistatin-related gene (FLRG). In: Protein Expression and Purification. 2006 ; Vol. 49, No. 1. pp. 78-82.
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Purification of recombinant activin A using the second follistatin domain of follistatin-related gene (FLRG). / Arai, Koji Y.; Tsuchida, Kunihiro; Li, Chun Mei; Watanabe, Gen; Sugino, Hiromu; Taya, Kazuyoshi; Nishiyama, Toshio.

In: Protein Expression and Purification, Vol. 49, No. 1, 01.01.2006, p. 78-82.

Research output: Contribution to journalArticle

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AU - Arai, Koji Y.

AU - Tsuchida, Kunihiro

AU - Li, Chun Mei

AU - Watanabe, Gen

AU - Sugino, Hiromu

AU - Taya, Kazuyoshi

AU - Nishiyama, Toshio

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N2 - Activins are multifunctional growth factors belonging to the transforming growth factor-β 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 βA-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.

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