TitleAggregation of ALS mutant superoxide dismutase expressed in Escherichia coli.
Publication TypeJournal Article
Year of Publication2004
AuthorsLeinweber B, Barofsky E, Barofsky DF, Ermilov V, Nylin K, Beckman JS
JournalFree Radic Biol Med
Date Published2004 Apr 01
KeywordsAcetylation, 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.

Alternate JournalFree Radic. Biol. Med.
PubMed ID15019975
Grant ListP01 ES00040 / ES / NIEHS NIH HHS / United States
P30 ES0021 / ES / NIEHS NIH HHS / United States
R01 NS033291 / NS / NINDS NIH HHS / United States