RRC ID 42497
Author Okumura S, Saitoh H, Wasano N, Katayama H, Higuchi K, Mizuki E, Inouye K.
Title Efficient solubilization, activation, and purification of recombinant Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli.
Journal Protein Expr. Purif.
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 microg/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 degrees C for 360 min.
Volume 47(1)
Pages 144-51
Published 2006-5
DOI 10.1016/j.pep.2005.10.011
PII S1046-5928(05)00356-6
PMID 16307894
MeSH Bacterial Proteins / chemistry Bacterial Proteins / genetics Bacterial Proteins / isolation & purification* Bacterial Proteins / toxicity Bacterial Toxins / chemistry Bacterial Toxins / genetics Bacterial Toxins / isolation & purification* Bacterial Toxins / toxicity Caco-2 Cells Chromatography, Ion Exchange Cloning, Molecular Endotoxins / chemistry Endotoxins / genetics Endotoxins / isolation & purification* Endotoxins / toxicity Escherichia coli / genetics* Hemolysin Proteins / chemistry Hemolysin Proteins / genetics Hemolysin Proteins / isolation & purification* Hemolysin Proteins / toxicity Humans Inclusion Bodies / chemistry* Inclusion Bodies / genetics* Inclusion Bodies / metabolism Inclusion Bodies / physiology Molecular Sequence Data Recombinant Proteins / chemistry Recombinant Proteins / genetics* Recombinant Proteins / isolation & purification* Recombinant Proteins / toxicity Solubility
IF 1.338
Times Cited 10
WOS Category BIOTECHNOLOGY & APPLIED MICROBIOLOGY BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Human and Animal Cells