Efficient solubilization, activation, and purification of recombinant Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli

Shiro Okumura, Hiroyuki Saitoh, Naoya Wasano, Hideki Katayama, Kazuhiko Higuchi, Eiichi Mizuki, Kuniyo Inouye

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

Abstract

A cytotoxic protein Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli was solubilized in 10 mM HCl. Protein concentration of saturated solution of the recombinant Cry45Aa in 10 mM HCl was about 25 times higher than that in the buffer of previous method (in 50 mM sodium carbonate buffer, pH 10.5, containing 1 mM EDTA, and 10 mM dithiothreitol). The Cry45Aa solubilized in the acidic solution was activated by pepsin as an alternative to proteinase K in the previous method. Cytotoxic activity against CACO-2 cells of the pepsin-treated Cry45Aa was almost identical to the proteinase K-treated protein. The pepsin-treated Cry45Aa was purified by cation-exchange chromatography. The concentration of the purified protein was 539 μg/ml, which was 27-fold higher than that of the activated Cry45Aa by the previously method. The cytotoxic activity of the purified protein was stable in broad pH region (pH 2.0-11.0) for 3 days, and 97% cytotoxic activity remained after incubation at 30 °C for 360 min.

Original languageEnglish
Pages (from-to)144-151
Number of pages8
JournalProtein Expression and Purification
Volume47
Issue number1
DOIs
Publication statusPublished - May 2006
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biotechnology

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