Expression of N-Terminal Cysteine Aβ 42 and Conjugation to Generate Fluorescent and Biotinylated Aβ 42 .

Autor:	Zhang S; Department of Chemistry, University of California-Irvine, Irvine, California 92697-2025, United States., Guaglianone G; Department of Chemistry, University of California-Irvine, Irvine, California 92697-2025, United States., Morris MA; Department of Chemistry, University of California-Irvine, Irvine, California 92697-2025, United States., Yoo S; Department of Chemistry, University of California-Irvine, Irvine, California 92697-2025, United States., Howitz WJ; Department of Chemistry, University of California-Irvine, Irvine, California 92697-2025, United States., Xing L; Irvine Materials Research Institute (IMRI), University of California-Irvine, Irvine, California 92697-2575, United States., Zheng JG; Irvine Materials Research Institute (IMRI), University of California-Irvine, Irvine, California 92697-2575, United States., Jusuf H; Department of Chemistry, University of California-Irvine, Irvine, California 92697-2025, United States., Huizar G; Department of Chemistry, University of California-Irvine, Irvine, California 92697-2025, United States., Lin J; Department of Chemistry, University of California-Irvine, Irvine, California 92697-2025, United States., Kreutzer AG; Department of Chemistry, University of California-Irvine, Irvine, California 92697-2025, United States., Nowick JS; Department of Chemistry, University of California-Irvine, Irvine, California 92697-2025, United States.; Department of Pharmaceutical Sciences, University of California-Irvine, Irvine, California 92697-2025, United States.
Jazyk:	angličtina
Zdroj:	Biochemistry [Biochemistry] 2021 Apr 20; Vol. 60 (15), pp. 1191-1200. Date of Electronic Publication: 2021 Apr 01.
DOI:	10.1021/acs.biochem.1c00105
Abstrakt:	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 42 with an N-terminal cysteine residue, Aβ (C1-42) , and its conjugation to generate Aβ 42 bearing fluorophores or biotin. The methods rely on the hitherto unrecognized observation that expression of the Aβ (MC1-42) gene yields the Aβ (C1-42) peptide, because the N-terminal methionine is endogenously excised by Escherichia coli . Conjugation of Aβ (C1-42) with maleimide-functionalized fluorophores or biotin affords the N-terminally labeled Aβ 42 . 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.
Databáze:	MEDLINE
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