A Robust And Efficient Production And Purification Procedure Of Recombinant Alzheimers Disease Methionine-Modified Amyloid-Beta Peptides
PLOS ONE(2016)
摘要
An improved production and purification method for Alzheimer's disease related methionine-modified amyloid-beta 1-40 and 1-42 peptides is proposed, taking advantage of the formation of inclusion body in Escherichia coli. A Thioflavin-S assay was set-up to evaluate inclusion body formation during growth and optimize culture conditions for amyloid-beta peptides production. A simple and fast purification protocol including first the isolation of the inclusion bodies and second, two cycles of high pH denaturation/neutralization combined with an ultrafiltration step on 30-kDa cut-off membrane was established. Special attention was paid to purity monitoring based on a rational combination of UV spectrophotometry and SDS-PAGE analyses at the various stages of the process. It revealed that this chromatography-free protocol affords good yield of high quality peptides in term of purity. The resulting peptides were fully characterized and are appropriate models for highly reproducible in vitro aggregation studies.
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关键词
peptides,purification procedure,methionine-modified
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