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Expression of N-Terminal Cysteine Aβ and Conjugation to Generate Fluorescent and Biotinylated Aβ. | LitMetric

AI Article Synopsis

Article Abstract

Fluorescent derivatives of the β-amyloid peptides (Aβ) are valuable tools for studying the interactions of Aβ with cells. Facile access to labeled expressed Aβ offers the promise of Aβ with greater sequence and stereochemical integrity, without impurities from amino acid deletion and epimerization. Here, we report methods for the expression of Aβ with an N-terminal cysteine residue, Aβ, and its conjugation to generate Aβ bearing fluorophores or biotin. The methods rely on the hitherto unrecognized observation that expression of the Aβ gene yields the Aβ peptide, because the N-terminal methionine is endogenously excised by . Conjugation of Aβ with maleimide-functionalized fluorophores or biotin affords the N-terminally labeled Aβ. The expression affords ∼14 mg of N-terminal cysteine Aβ from 1 L of bacterial culture. Subsequent conjugation affords ∼3 mg of labeled Aβ from 1 L of bacterial culture with minimal cost for labeling reagents. High-performance liquid chromatography analysis indicates the N-terminal cysteine Aβ to be >97% pure and labeled Aβ peptides to be 94-97% pure. Biophysical studies show that the labeled Aβ peptides behave like unlabeled Aβ and suggest that labeling of the N-terminus does not substantially alter the properties of the Aβ. We further demonstrate applications of the fluorophore-labeled Aβ peptides by using fluorescence microscopy to visualize their interactions with mammalian cells and bacteria. We anticipate that these methods will provide researchers convenient access to useful N-terminally labeled Aβ, as well as Aβ with an N-terminal cysteine that enables further functionalization.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9059633PMC
http://dx.doi.org/10.1021/acs.biochem.1c00105DOI Listing

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