Aggregation of ALS mutant superoxide dismutase expressed in Escherichia coli.
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.
Leinweber
Barbara
B
Linus Pauling Institute, Environmental Health Sciences Center, 1011 ALS, Oregon State University, Corvallis, OR 97331-7302, USA.
Barofsky
Elisabeth
E
Barofsky
Douglas F
DF
Ermilov
Vladimir
V
Nylin
Keith
K
Beckman
Joseph S
JS
D000107:Acetylation; D019943:Amino Acid Substitution; D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D008623:Mercaptoethanol / Q000737:chemistry; D020125:Mutation, Missense; D018272:Porins / Q000032:analysis / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry
C109237:OmpF protein; D018272:Porins; D011994:Recombinant Proteins; D008623:Mercaptoethanol; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc