title: Novel semisynthetic method for generating full length beta-amyloid peptides.
abstract: Bacterial expression of full length beta-amyloid (Abeta) is problematic because of toxicity and poor solubility of the expressed protein, and a strong tendency of Met35 to become oxidized in inclusion bodies. We have developed a semisynthetic method in which Abeta1-29 is expressed in bacteria as part of a fusion protein with a C-terminal intein and Chitin-Binding Domain (CBD). There is also a single residue, N-terminal Met extension. The protein, Met-Abeta1-29-Intein-CBD, is well expressed and highly water-soluble. After binding of the expressed protein to Chitin beads, treatment with sodium 2-mercapto-ethane sulfonate (MESNA) yields Met-Abeta1-29-MESNA, with a C-terminal thioester suitable for native chemical ligation. Met-Abeta1-29-MESNA is first subjected to CNBr cleavage, which removes the N-terminal Met residue, but leaves the thioester intact. We synthesized NH2-A30C-Abeta30-40, which has an N-terminal Cys residue and is the partner for native chemical ligation with Met-Abeta1-29-MESNA. Native chemical ligation proceeds rapidly and efficiently (>90% yield) to give A30C-Abeta1-40. The final step is selective desulfurization using Raney-Ni, which also proceeds rapidly and efficiently (>90% yield) to give native sequence Abeta1-40. Overall, this system is highly efficient, and can yield approximately 8-10 mg of pure Abeta1-40 from one liter of bacterial culture medium. This procedure is adaptable for producing other Abeta peptides. We have also expressed an Abeta construct bearing a point mutation associated with one type of familial Alzheimer's Disease, the Iowa mutation, i.e., Met-D23N-Abeta1-29-Intein-CBD. Since expression of the intein-containing fusion protein is robust in minimal media as well as standard enriched media, this procedure also can be readily modified for incorporating 15N or 13C labels for NMR. Future work will also include extending this system to longer Abeta peptides, such as Abeta1-42.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
authors.forename: Jessica J
authors.initials: JJ
authors.lastname: Bockhorn
authors.forename: Kristi L
authors.initials: KL
authors.lastname: Lazar
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Gasser
authors.forename: Laura M
authors.initials: LM
authors.lastname: Luther
authors.forename: Isam M
authors.initials: IM
authors.lastname: Qahwash
authors.forename: Neeraj
authors.initials: N
authors.lastname: Chopra
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Meredith
mesh_terms: D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000096:biosynthesis* / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D016229:Amyloid beta-Peptides; D010455:Peptides; D011994:Recombinant Proteins; D002686:Chitin