Total Chemical Synthesis of Crambin

Bang, Duhee; Chopra, Neeraj; Kent, Stephen B. H.

doi:10.1021/ja0385078

Cited by 99 publications

(85 citation statements)

References 21 publications

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“…Total chemical synthesis of proteins offers unlimited versatility regarding the type, number, and localization of unnatural aminoacids to be introduced. 10 Here, we present the first report of an efficient one-pot total chemical synthesis of a-syn and demonstrate that synthetic a-syn exhibits similar biophysical, membrane binding and aggregation properties to recombinant a-syn obtained from E. coli.…”

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confidence: 76%

One-pot total chemical synthesis of human α-synuclein

et al. 2013

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confidence: 76%

One-pot total chemical synthesis of human α-synuclein

et al. 2013

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“…[50] The assembly of larger proteins, however, requires the development of a multistep ligation procedure which can be rather technically difficult. The combination of chemical ligation and biosynthetic methods therefore is an attractive strategy to construct proteins of, in principle, unlimited size and of designed composition.…”

Section: Combination Of Chemical Ligation and Biosynthetic Methodsmentioning

confidence: 99%

Diels–Alder Ligation of Peptides and Proteins

Araújo

Palomo

Cramer

et al. 2006

Chemistry A European J

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“…We have previously developed synthetic routes to the protein crambin that involve the ligation of three unprotected peptide segments. Chemical ligation of the peptide segments and the folding of the full-length polypeptide have been highly optimized (30,32). Thus, crambin was chosen as a suitable target with which to examine His 6 tag-assisted chemical protein synthesis.…”

Section: Resultsmentioning

confidence: 99%

His₆tag-assisted chemical protein synthesis

Bang

Kent

2005

Proc. Natl. Acad. Sci. U.S.A.

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To make more practical the total chemical synthesis of proteins by the ligation of unprotected peptide building blocks, we have developed a method to facilitate the isolation and handling of intermediate products. The synthetic technique makes use of a His6 tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a His6 tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis. We used syntheses of crambin and a modular tetratricopeptide repeat protein of 17 kDa as models to examine the utility of this affinity purification approach. The results show that His6 tag-assisted chemical protein synthesis is a useful method that substantially reduces handling losses and provides for rapid chemical protein syntheses. (16). Synthetic access to protein molecules has been used to elucidate the molecular basis of protein folding and stability (17), to elucidate the molecular basis of protein function (18), to design and build proteins of novel structure (19), and to determine the molecular structure of proteins by both NMR (20) and x-ray crystallography (21). Chemical protein synthesis has also been used to develop candidate protein therapeutic molecules with improved properties (21,22).Thus far, most research on chemically synthesized protein molecules (16) has been focused on relatively small proteins in the size range of 50 to Ϸ150 aa (i.e., made from two or three peptide segments). This size limitation is the result of two phenomena: (i) practical constraints on the size of the unprotected peptide segment building blocks (23) and (ii) technical challenges in the chemical ligation of more than two or three peptide segments. Even highly optimized stepwise solid-phase peptide synthetic procedures are limited to Ϸ50 amino acid residues for the practical preparation of high-purity unprotected peptides (24). Thus, the chemical synthesis of a protein of typical size (Ϸ300 aa) (25) would require the use of at least six synthetic peptide segments as building blocks.The most effective way of covalently joining unprotected peptide segments to form a protein molecule is native chemical ligation (3,26). Native chemical ligation involves the reaction of a peptide-␣ thioester with a Cys-peptide; reversible thioesterthiol exchange with the N-terminal Cys residue gives a thioesterlinked intermediate that undergoes an irreversible intramolecular rearrangement to give a near-quantitative yield of a single product linked by a native peptide bond at the ligation site. The native chemical ligation reaction is both practical and highly effective. ¶ Each ligation product can be purified by reverse-phase HPLC and characterized with great precision by electrospray M...

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Total Chemical Synthesis of Crambin

Cited by 99 publications

References 21 publications

One-pot total chemical synthesis of human α-synuclein

One-pot total chemical synthesis of human α-synuclein

Diels–Alder Ligation of Peptides and Proteins

His₆tag-assisted chemical protein synthesis

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Total Chemical Synthesis of Crambin

Cited by 99 publications

References 21 publications

One-pot total chemical synthesis of human α-synuclein

One-pot total chemical synthesis of human α-synuclein

Diels–Alder Ligation of Peptides and Proteins

His6tag-assisted chemical protein synthesis

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His₆tag-assisted chemical protein synthesis