Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis

Shunichi Akiba, Yasuhiro Hayashi, Yoshihiro Hakamada, Keiji Endo, Katsutoshi Ara, Shuji Kawai, Eiichi Saitoh

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

We herein describe the development of a Bacillus subtilis system that can be used to produce large quantities of recombinant (r-) human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily. The B. subtilis that lacked the alkaline protease E gene (ΔaprE type mutant strain) was prepared by homologous recombination. The cDNA fragments coding for mature cystatins (S and SA) were ligated in frame to the DNA segment for the signal peptide of endoglucanase in the pHSP-US plasmid vector that was then use to transform the ΔaprE type mutant strain of B. subtilis. The transformants carrying the expression vectors were cultivated in 5-L jar fermenters for 3 days at 30 °C. Both r-cystatin S and r-cystatin SA were successfully expressed and secreted into the culture broth, and were purified using a fast performance liquid chromatography system. The first use of ΔaprE type mutant strain of B. subtilis made it possible to obtain a high yield of secreted protein, which makes this system an improvement over expression in Escherichia coli. We conclude that this system has high utility for expression of commercial quantities of secreted proteins.

Original languageEnglish
Pages (from-to)203-210
Number of pages8
JournalProtein Expression and Purification
Volume49
Issue number2
DOIs
Publication statusPublished - 2006 Oct 1
Externally publishedYes

Fingerprint

Salivary Cystatins
Bacillus subtilis
Cystatins
Cysteine Proteinase Inhibitors
Cellulase
Homologous Recombination
Protein Sorting Signals
Liquid Chromatography
Proteins
Plasmids
Complementary DNA
Escherichia coli
DNA
Genes

Keywords

  • Bacillus subtilis
  • Endoglucanase
  • Human salivary cystatin

ASJC Scopus subject areas

  • Biotechnology

Cite this

Akiba, S., Hayashi, Y., Hakamada, Y., Endo, K., Ara, K., Kawai, S., & Saitoh, E. (2006). Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis. Protein Expression and Purification, 49(2), 203-210. https://doi.org/10.1016/j.pep.2006.04.005

Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis. / Akiba, Shunichi; Hayashi, Yasuhiro; Hakamada, Yoshihiro; Endo, Keiji; Ara, Katsutoshi; Kawai, Shuji; Saitoh, Eiichi.

In: Protein Expression and Purification, Vol. 49, No. 2, 01.10.2006, p. 203-210.

Research output: Contribution to journalArticle

Akiba, S, Hayashi, Y, Hakamada, Y, Endo, K, Ara, K, Kawai, S & Saitoh, E 2006, 'Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis', Protein Expression and Purification, vol. 49, no. 2, pp. 203-210. https://doi.org/10.1016/j.pep.2006.04.005
Akiba, Shunichi ; Hayashi, Yasuhiro ; Hakamada, Yoshihiro ; Endo, Keiji ; Ara, Katsutoshi ; Kawai, Shuji ; Saitoh, Eiichi. / Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis. In: Protein Expression and Purification. 2006 ; Vol. 49, No. 2. pp. 203-210.
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