Synthesis of theC-terminal region of opioid receptor like 1 in an SDS micelle by the native chemical ligation: effect of thiol additive and SDS concentration on ligation efficiency

Sato, Takeshi; Saito, Yasuhiro; Aimoto, Saburo

doi:10.1002/psc.634

Cited by 72 publications

(85 citation statements)

References 22 publications

(28 reference statements)

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“…The first strategy was developed by Aimoto and co-workers, building upon earlier work by Deber [74] and Muir [75] on the synthesis of hydrophobic proteins. Aimoto and co-workers incorporated five Arg residues into the thiol moiety of the thioester fragment [72]. This allowed them to synthesize the C-terminal 83 residues of ORL1 (opioid receptor-like 1).…”

Section: Methods For the Synthesis Of Long/difficult Peptidesmentioning

confidence: 99%

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New Methods for Chemical Protein Synthesis

Guan

Chaffey

Zeng

2014

Topics in Current Chemistry

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Chemical protein synthesis is a useful tool to generate pure proteins which are otherwise difficult to obtain in sufficient amounts for structure and property analysis. Additionally, because of the precise and flexible nature of chemical synthesis, it allows for controllable variation of protein sequences, which is valuable for understanding the relationships between protein structure and function. Despite the usefulness of chemical protein synthesis, it has not been widely adopted as a tool for protein characterization, mainly because of the lack of general and efficient methods for the preparation and coupling of peptide fragments and for the folding of polypeptide chains. To address these issues, many new methods have recently been developed in the areas of solid-phase peptide synthesis, peptide fragment assembly, and protein folding. Here we review these recent technological advances and highlight the gaps needing to be addressed in future research.

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Section: Methods For the Synthesis Of Long/difficult Peptidesmentioning

confidence: 99%

“…A couple of strategies were also developed to increase the solubility and/or decrease the aggregation propensity of peptides once cleaved from solid supports [63,72,73]. The first strategy relies upon the use of a C-terminal (Arg) 5/6 to increase the solubility of peptide fragments (Fig.…”

Section: Methods For the Synthesis Of Long/difficult Peptidesmentioning

confidence: 99%

New Methods for Chemical Protein Synthesis

Guan

Chaffey

Zeng

2014

Topics in Current Chemistry

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“…Several groups have addressed this challenge by designing clever chemical methods for temporarily improving handling properties. The solubility of initial peptide segments can be improved by incorporation of pH-sensitive isoacyl dipeptide building blocks 49 (at Ser/Thr residues) or application of the thioester Arg n tag strategy 50,51 . Danishefsky’s group employed custom Glu and Lys building blocks equipped with allylic ester and allylic carbamate linkers containing solubilizing guanidine groups 52 .…”

Section: Introductionmentioning

confidence: 99%

Aligator: A computational tool for optimizing total chemical synthesis of large proteins

Jacobsen

Erickson

Kay

2017

Bioorganic & Medicinal Chemistry

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The scope of chemical protein synthesis (CPS) continues to expand, driven primarily by advances in chemical ligation tools (e.g., reversible solubilizing groups and novel ligation chemistries). However, the design of an optimal synthesis route can be an arduous and fickle task due to the large number of theoretically possible, and in many cases problematic, synthetic strategies. In this perspective, we highlight recent CPS tool advances and then introduce a new and easy-to-use program, Aligator (Automated Ligator), for analyzing and designing the most efficient strategies for constructing large targets using CPS. As a model set, we selected the E. coli ribosomal proteins and associated factors for computational analysis. Aligator systematically scores and ranks all feasible synthetic strategies for a particular CPS target. The Aligator script methodically evaluates potential peptide segments for a target using a scoring function that includes solubility, ligation site quality, segment lengths, and number of ligations to provide a ranked list of potential synthetic strategies. We demonstrate the utility of Aligator by analyzing three recent CPS projects from our lab: TNFα (157 aa), GroES (97 aa), and DapA (312 aa). As the limits of CPS are extended, we expect that computational tools will play an increasingly important role in the efficient execution of ambitious CPS projects such as production of a mirror-image ribosome.

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“…For example, oxidation of Met35 was employed to suppress aggregation of resin bound Aβ 42 during synthesis, followed by reduction after purification 42 . Reversible solubilization has also been accomplished by adding, during the synthesis, a cationic C-terminus separated from the peptide of interest by a labile non-peptide bond, which can either be removed to directly generate the peptide of interest 43,44 , or can be incorporated into a fragment condensation approach to make longer polypeptides 45–47 . Data on the efficiencies of cationic tail removal are generally not reported, however, making it difficult to assess the efficacy of this method.…”

Section: Introductionmentioning

confidence: 99%

Improved chemical synthesis of hydrophobic Aβ peptides using addition of C‐terminal lysines later removed by carboxypeptidase B

2014

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Many amyloidogenic peptides are highly hydrophobic, introducing significant challenges to obtaining high quality peptides by chemical synthesis. For example, while good yield and purity can be obtained in the solid phase synthesis of the Alzheimer’s plaque peptide Aβ40, addition of a C-terminal Ile-Ala sequence to generate the more toxic Aβ42 molecule creates a much more difficult synthesis resulting in low yields and purities. We describe here a new method that significantly improves the Fmoc solid phase synthesis of Aβ peptides. In our method, Lys residues are linked to the desired peptide’s C-terminus through standard peptide bonds during the synthesis. These Lys residues are then removed post-purification using immobilized carboxypeptidase B. With this method we obtained both Aβ42 and Aβ46 of superior quality that, for Aβ42, rivals that obtained by recombinant expression. Intriguingly, the method appears to provide independent beneficial effects on both the total synthetic yield and on purification yield and final purity. Reversible Lys addition with carboxypeptidase B removal should be a generally useful method for making hydrophobic peptides that is applicable to any sequence not ending in Arg or Lys. As expected from the additional hydrophobicity of Aβ46, which is extended from the sequence Aβ42 by a C-terminal Thr-Val-Ile-Val sequence, this peptide makes typical amyloid at rates significantly faster than for Aβ42 or Aβ40. The enhanced amyloidogenicity of Aβ46 suggests that, even though it is present in relatively low amounts in the human brain, it could play a significant role in helping to initiate Aβ amyloid formation.

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Synthesis of theC-terminal region of opioid receptor like 1 in an SDS micelle by the native chemical ligation: effect of thiol additive and SDS concentration on ligation efficiency

Cited by 72 publications

References 22 publications

New Methods for Chemical Protein Synthesis

New Methods for Chemical Protein Synthesis

Aligator: A computational tool for optimizing total chemical synthesis of large proteins

Improved chemical synthesis of hydrophobic Aβ peptides using addition of C‐terminal lysines later removed by carboxypeptidase B

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