|Aggregation of ALS mutant superoxide dismutase expressed in Escherichia coli.
|Year of Publication
|Leinweber B, Barofsky E, Barofsky DF, Ermilov V, Nylin K, Beckman JS
|Free Radic Biol Med
|2004 Apr 01
|Acetylation, Amino Acid Substitution, Amyotrophic Lateral Sclerosis, Cloning, Molecular, Copper, Escherichia coli, Gene Expression, Humans, Inclusion Bodies, Mercaptoethanol, Mutation, Missense, Porins, Protein Folding, Recombinant Proteins, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Superoxide Dismutase, Zinc
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 degrees C, but >95% A4V SOD aggregated in inclusion bodies when expressed at 23 degrees 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 beta-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.
|Free Radic. Biol. Med.
|P01 ES00040 / ES / NIEHS NIH HHS / United States
P30 ES0021 / ES / NIEHS NIH HHS / United States
R01 NS033291 / NS / NINDS NIH HHS / United States