| 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 |
2006-5-1
|
| DOI |
10.1016/j.pep.2005.10.011
|
| PII |
S1046-5928(05)00356-6
|
| PMID |
16307894
|
| MeSH |
Bacillus thuringiensis Toxins
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 |
1.513
|
| Times Cited |
14
|
|
WOS Category
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
BIOCHEMICAL RESEARCH METHODS
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| Resource |
| Human and Animal Cells |
CACO-2(RCB0988) |
