Title of article :
Aggregation of ALS mutant superoxide dismutase expressed in Escherichia coli
Author/Authors :
Barbara Leinweber، نويسنده , , Elisabeth Barofsky، نويسنده , , Douglas F. Barofsky، نويسنده , , Vladimir Ermilov، نويسنده , , Keith Nylin، نويسنده , , Joseph S. Beckman، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2004
Abstract :
Although large amounts of wild-type human Cu,Zn superoxide dismutase (SOD) are easily expressed in Escherichia coli, the amyotrophic lateral sclerosis-associated mutants have a strong propensity to aggregate into inclusion bodies. The alanine to valine mutation at the fourth codon (A4V) is responsible for a rapidly progressive disease course and is particularly prone to aggregation when expressed in E. coli. We found that A4V SOD remained soluble when expressed at 18°C, but >95% A4V SOD aggregated in inclusion bodies when expressed at 23°C or above. The SOD aggregates dissolved with 4 M urea, suggesting that intermolecular hydrophobic interactions were predominantly responsible for making SOD insoluble. Many of the urea-solubilized subunits were cross-linked via disulfide bridges. Fully active mutant SOD could be produced by dialyzing urea away in the presence of β-mercaptoethanol and subsequently adding copper plus zinc, providing a fast procedure for purifying hundreds of milligrams of protein. Extensive rinsing removed most contaminating E. coli proteins from A4V SOD inclusion bodies except for a 37 kDa protein identified as outer membrane protein F using MALDI ToF/ToF mass spectrometry. Our results indicate that metal-deficient ALS-mutant SOD folds into stable apo conformation able to rebind metals. At high protein concentrations, SOD forms aggregates through hydrophobic interactions between subunits that seem to act as a kinetic snare to entrap additional proteins.
Keywords :
superoxide dismutase , Lou Gehrigיs disease , ALS , copper , zinc , free radicals , aggregation
Journal title :
Free Radical Biology and Medicine
Journal title :
Free Radical Biology and Medicine