Dataset Information

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.

SUBMITTER: Bockhorn JJ

PROVIDER: S-EPMC2965776 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Novel semisynthetic method for generating full length beta-amyloid peptides.

Bockhorn Jessica J JJ Lazar Kristi L KL Gasser Adam J AJ Luther Laura M LM Qahwash Isam M IM Chopra Neeraj N Meredith Stephen C SC

Biopolymers 20100101 4

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 h ...[more]

PMID: 20593467

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Project description:IntroductionWhole-metagenome sequencing can be a rich source of information about the structure and function of entire metagenomic communities, but getting accurate and reliable results from these datasets can be challenging. Analysis of these datasets is founded on the mapping of sequencing reads onto known genomic regions from known organisms, but short reads will often map equally well to multiple regions, and to multiple reference organisms. Assembling metagenomic datasets prior to mapping can generate much longer and more precisely mappable sequences but the presence of closely related organisms and highly conserved regions makes metagenomic assembly challenging, and some regions of particular interest can assemble poorly. One solution to these problems is to use specialised tools, such as Kelpie, that can accurately extract and assemble full-length sequences for defined genomic regions from whole-metagenome datasets.MethodsKelpie is a kMer-based tool that generates full-length amplicon-like sequences from whole-metagenome datasets. It takes a pair of primer sequences and a set of metagenomic reads, and uses a combination of kMer filtering, error correction and assembly techniques to construct sets of full-length inter-primer sequences.ResultsThe effectiveness of Kelpie is demonstrated here through the extraction and assembly of full-length ribosomal marker gene regions, as this allows comparisons with conventional amplicon sequencing and published metagenomic benchmarks. The results show that the Kelpie-generated sequences and community profiles closely match those produced by amplicon sequencing, down to low abundance levels, and running Kelpie on the synthetic CAMI metagenomic benchmarking datasets shows similar high levels of both precision and recall.ConclusionsKelpie can be thought of as being somewhat like an in-silico PCR tool, taking a primer pair and producing the resulting 'amplicons' from a whole-metagenome dataset. Marker regions from the 16S rRNA gene were used here as an example because this allowed the overall accuracy of Kelpie to be evaluated through comparisons with other datasets, approaches and benchmarks. Kelpie is not limited to this application though, and can be used to extract and assemble any genomic region present in a whole metagenome dataset, as long as it is bound by a pairs of highly conserved primer sequences.

Dataset Information

Novel semisynthetic method for generating full length beta-amyloid peptides.

Publications

Novel semisynthetic method for generating full length beta-amyloid peptides.

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