Solid phase chemical ligation employing a rink amide linker for the synthesis of histone H2B protein

Jbara, Muhammad; Seenaiah, Mallikanti; Brik, Ashraf

doi:10.1039/c4cc06499b

Cited by 44 publications

(43 citation statements)

References 28 publications

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“…Establishing such solid-phase chemistry for connecting amino acids underpinned the breakthroughs in the biological understanding and therapeutic use of peptides (14,15), whereas the solid-phase synthesis of DNA primers underpinned the revolution in gene amplification and reengineering (16,17). Solid-phase reaction has also enabled the ligation of peptide fragments to make synthetic proteins (18,19).…”

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confidence: 99%

Programmable polyproteams built using twin peptide superglues

Veggiani

Nakamura

Brenner

et al. 2016

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

294

360

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Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Here we achieved sequenceprogrammed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. SpyTag peptide is engineered to spontaneously form an isopeptide bond with SpyCatcher protein. By engineering the adhesin RrgA from Streptococcus pneumoniae, we developed the peptide SnoopTag, which formed a spontaneous isopeptide bond to its protein partner SnoopCatcher with >99% yield and no crossreaction to SpyTag/SpyCatcher. Solid-phase attachment followed by sequential SpyTag or SnoopTag reaction between building-blocks enabled iterative extension. Linear, branched, and combinatorial polyproteins were synthesized, identifying optimal combinations of ligands against death receptors and growth factor receptors for cancer cell death signal activation. This simple and modular route to programmable "polyproteams" should enable exploration of a new area of biological space.synthetic biology | protein engineering | nanobiotechnology | split protein | antibody

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mentioning

confidence: 99%

Programmable polyproteams built using twin peptide superglues

Veggiani

Nakamura

Brenner

et al. 2016

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

294

360

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“…23, 24 In order to minimize the number of segments, one-pot approaches require long peptide sequences resulting in challenging syntheses. 21 Solid phase ligation (SP-NCL) has been reported to improve efficiency and yield for histone H2B, 22 but we show in this work that for H4 and CpA, overall yields with SP-NCL are unacceptably low despite efficient chemistry at each step. Here, we demonstrate that carrying out initial ligation steps on the solid phase followed by final ligation steps in solution provides optimal efficiency and yield for heavily modified synthetic histone proteins, contrary to previous reports that suggest fully solid phase ligation is inherently superior to mixed phase in other systems.…”

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confidence: 71%

“…22 Each approach has both advantages and disadvantages. Sequential approaches require intermediary purification steps, which drastically decrease overall yields.…”

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confidence: 99%

“…25, 28, 29 For histone, Brik et al recently used the TFA-labile Rink linker to prepare H2B, which results in the conservative substitution of an amide at the C-terminus. 22 However, for proteins such as H4 and CpA, for which the carboxyl termini are each folded into the core of the nucleosome and are positioned to participate in salt bridge interactions, 30 any chemical modification at this site is not appropriate. We therefore developed a dual-linker strategy in which the C-terminal residue is followed by an internal ligation-stable HMBA linker 31 and a ligation handle, which allows for sequence-independent resin attachment and generates the native carboxyl terminus in a simple pH shift in aqueous mixtures.…”

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confidence: 99%

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Hybrid phase ligation for efficient synthesis of histone proteins

Mahto

Justus

et al. 2016

Org. Biomol. Chem.

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We introduce a hybrid solid-solution phase ligation approach that combines the efficiency of solid phase ligation with solution phase ligation in the total synthesis of modified histone proteins. A two linker strategy allows analysis throughout work on the solid phase and maximizes yields through cleavage at an external Rink, while an internal HMBA linker allows the native carboxyl terminus for any protein sequence. We demonstrate this approach for two histone proteins: triple-acetylated H4-K5ac,K12ac,K91ac and CENP-A-K124ac.

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“…In addition to summing the individual scores from each segment, ligation strategies receive an additional score based on the number of ligations. For traditional NCL (excluding various one-pot and solid-phase approaches, e.g., 78,79 ), each ligation step adds an HPLC purification step that ultimately reduces yield. To discourage strategies that have more than an ideal number of ligations, this scoring function penalizes strategies having an average segment length <40 residues (−2 for each “excessive” ligation beyond this limit).…”

Section: Discussionmentioning

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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Solid phase chemical ligation employing a rink amide linker for the synthesis of histone H2B protein

Cited by 44 publications

References 28 publications

Programmable polyproteams built using twin peptide superglues

Programmable polyproteams built using twin peptide superglues

Hybrid phase ligation for efficient synthesis of histone proteins

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

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