Princeton University

The Rockefeller University

The Scripps Research Institute

Other

New York University

Tom W Muir

Neel H Shah

Stephen B H Kent

Philip E Dawson

Ronald Beavis

Richard P Novick

Guangyong Ji

Miquel Vila-Perello

Jung-Min Kee

Zhihua Liu

Patricia Mayville

Manuel M Mueller

Adam J Stevens

Rob C Oslund

Lynne Canne Bannen

Philip A Cole

Steven J Bark

Dolan Sondhi

Konstantine Severinov

Michael C Fitzgerald

Uyen T T Nguyen

Embodiments of the present invention relate to inteins, split inteins, compositions comprising inteins and methods for use of these.

Split inteins with exceptional splicing activity

Provided are phosphonopyrazole-based phosphohistidine analogs that are useful as haptens for the preparation of immunogens, immunogens that include these haptens linked to carrier molecules, antibodies thereto and uses of these antibodies, haptens, immunogens and phosphohistidine analogs.

Phosphohistidine mimetics and antibodies to same

Disclosed herein are split inteins, fused proteins of split inteins, and methods of using split inteins to efficiently purify and modify proteins of interest.

Split inteins, conjugates and uses thereof

Compositions and methods are provided for DNA barcoding of designer mononucleosome and polynucleosome (chromatin array) libraries for use, for example, for the profiling of chromatin readers, writers, erasers, and modulators thereof.

DNA barcoding of designer mononucleosome and chromatin array libraries for the profiling of chromatin readers, writers, erasers, and modulators thereof

Method using split inteins to generate protein conjugates

Method of using split inteins, conjugates to generate protein conjugates

Provided are phosphonopyrazole-based phosphohistidine analogs that are useful as haptens for the preparation of immunogens, immunogens that include these haptens linked to carrier molecules, antibodies thereto and uses of these antibodies, haptens, immunogens and phosphohistidine analogs. The invention is based in part on new pHis analogs that are chemically stable and which have been used as haptens and immunogens to generate specific anti-pHis antibodies that bind with high affinity and specificity.

The present invention relates to the phosphohistidine analogs of the present invention which of the formula and the hapten containing the residue of same. It also relates to the hapten conjugated to a carrier molecule and the isolated antibodies raised against the immunogens, said antibodies recognizing polypeptide containing a phosphorylated histidine or the phosphotriazole residue but it does not recognize an amino acid or polypeptide that is not phosphorylated or a polypeptide which is phosphorylated on amino acids other than histidine but not on histidine.

Phosphohistidine analogs

The present invention relates to the phosphohistidine analogs of the present invention which of the formula (I) and the hapten containing the residue of same. It also relates to the hapten conjugated to a carrier molecule and the isolated antibodies raised against the immunogens, said antibodies recognizing polypeptide containing a phosphorylated histidine or the phosphotriazole residue but it does not recognize an amino acid or polypeptide that is not phosphorylated or a polypeptide which is phosphorylated on amino acids other than histidine but not on histidine. 

The present invention provides a cyclic peptide comprising the structure: wherein X is selected from the group consisting of an amino acid, an amino acid analog, a peptidomimetic and a non-amide isostere, Z is selected from the group consisting of a synthetic amino acid and a biosynthetic amino acid, R is selected from the group consisting of oxygen, nitrogen, sulfur and carbon, n is 0 to 10 and y is 1 to 10. The present invention also provides a cyclic peptide comprising the amino acid sequence of NH&#x2014;X-Z-X&#x2014;COOH and a cyclic bond between the Z residue and COOH other than a thioester bond, wherein X is selected from the group consisting of to an amino acid, an amino acid analog, a peptidomimetic and a non-amide isostere, Z is selected from the group consisting of a synthetic amino acid and a biosynthetic amino acid, n is 0 to 10 and y is 1 to 10. Methods of preparation including a cyclization protocol, and methods of use of the cyclic peptides of the invention are also disclosed.

[object Object]

Proteins of moderate size having native peptide backbones are produced by a method of native chemical ligation. Native chemical ligation employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The transient thioester-linked intermediate then spontaneously undergoes a rearrangement to provide the full length ligation product having a native peptide bond at the ligation site. Full length ligation products are chemically identical to proteins produced by cell free synthesis. Full length ligation products may be refolded and/or oxidized, as allowed, to form native disulfide-containing protein molecules. The technique of native chemical ligation is employable for chemically synthesizing full length proteins.

Synthesis of proteins by native chemical ligation

The present invention provides a cyclic peptide comprising the structure:wherein X is selected from the group consisting of an amino acid, an amino acid analog, a peptidomimetic and a non-amide isostere, Z is selected from the group consisting of a synthetic amino acid and a biosynthetic amino acid, R is selected from the group consisting of oxygen, nitrogen, sulfur and carbon, n is 0 to 10 and y is 1 to 10. The present invention also provides a cyclic peptide comprising the amino acid sequence of NH&#x2014;X-Z-X&#x2014;COOH and a cyclic bond between the Z residue and COOH other than a thioester bond, wherein X is selected from the group consisting of an amino acid, an amino acid analog, a peptidomimetic and a non-amide isostere, Z is selected from the group consisting of a synthetic amino acid and a biosynthetic amino acid, n is 0 to 10 and y is 1 to 10. Methods of preparation including a cyclization protocol, and methods of use of the cyclic peptides of the invention are also disclosed.

Staphylococcus peptides for bacterial interference

The present invention provides a method of cleaving a recombinantly expressed protein from an intein and ligating the protein to a peptide containing an N-terminal cysteine having an unoxidized sulfhydryl side chain which comprises contacting the protein with the peptide in a reaction solution comprising a conjugated thiophenol, thereby forming a C-terminal thioester of the recombinant protein which spontaneously rearranges intramolecularly to form an amide bond linking the protein to the peptide. The present invention also provides a method of producing a protein-chip composition comprising the steps of: (a) contacting a solid support chip containing an amine group with a peptide containing an N-terminal cysteine having an unoxidized sulfhydryl side chain thereby covalently linking the peptide to the solid support, forming a peptide-chip; and (b) contacting a recombinant protein having an intein domain with the peptide-chip of step (a) with a reaction solution containing a conjugated thiophenol, thereby forming a C-terminal thioester of the recombinant protein which spontaneously rearranges intramoleculaly to form an amide bond covalently linking the protein to the peptide-chip, thereby producing a protein-chip composition.

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Princeton, NJ, United States of America

Tom W Muir