Synthesis of covalent DNA–protein conjugates by expressed protein ligation

2005 ◽  
Vol 1 (1) ◽  
pp. 64 ◽  
Author(s):  
Marina Lovrinovic ◽  
Mark Spengler ◽  
Carl Deutsch ◽  
Christof M. Niemeyer
Keyword(s):  
Expressed Protein Ligation ◽  
Protein Ligation ◽  
Protein Conjugates

scholarly journals Expressed Protein Ligation Without Intein

2020 ◽  
Author(s):  
Yuchen Qiao ◽  
Ge Yu ◽  
Xiaoyan Wang ◽  
Kaci C. Kratch ◽  
Wesley Wei Wang ◽  
...  
Keyword(s):  
Rnase H ◽  
Protein Chemistry ◽  
Histone H2a ◽  
Expressed Protein Ligation ◽  
Protein Ligation ◽  
Protein Conjugates ◽  
Synthetic Capacity ◽  
New Research ◽  
Nucleophilic Acyl Substitution ◽  
Fusion Approach

Proteins with a functionalized <i>C</i>-terminus such as a <i>C</i>-terminal thioester are key to the synthesis of larger proteins via expressed protein ligation. They are usually made by recombinant fusion to intein. Although powerful, the intein fusion approach suffers from premature hydrolysis and low compatibility with denatured conditions. To totally bypass the involvement of an enzyme for expressed protein ligation, here we showed that a cysteine in a recombinant protein was chemically activated by a small molecule cyanylating reagent at its <i>N</i>-side amide for undergoing nucleophilic acyl substitution with amines including a number of L- and D-amino acids and hydrazine. The afforded protein hydrazides could be used further for expressed protein ligation. We demonstrated the versatility of this approach with the successful synthesis of ubiquitin conjugates, ubiquitin-like protein conjugates, histone H2A with a posttranslational modification, RNAse H that actively hydrolyzed RNA, and exenatide that is a commercial therapeutic peptide. The technique, which is exceedingly simple but highly useful, expands to a great extent the synthetic capacity of protein chemistry and will therefore make a large avenue of new research possible.

scholarly journals Expressed Protein Ligation Without Intein

2020 ◽  
Author(s):  
Yuchen Qiao ◽  
Ge Yu ◽  
Xiaoyan Wang ◽  
Kaci C. Kratch ◽  
Wesley Wei Wang ◽  
...  
Keyword(s):  
Rnase H ◽  
Protein Chemistry ◽  
Histone H2a ◽  
Expressed Protein Ligation ◽  
Protein Ligation ◽  
Protein Conjugates ◽  
Synthetic Capacity ◽  
New Research ◽  
Nucleophilic Acyl Substitution ◽  
Fusion Approach

Proteins with a functionalized <i>C</i>-terminus such as a <i>C</i>-terminal thioester are key to the synthesis of larger proteins via expressed protein ligation. They are usually made by recombinant fusion to intein. Although powerful, the intein fusion approach suffers from premature hydrolysis and low compatibility with denatured conditions. To totally bypass the involvement of an enzyme for expressed protein ligation, here we showed that a cysteine in a recombinant protein was chemically activated by a small molecule cyanylating reagent at its <i>N</i>-side amide for undergoing nucleophilic acyl substitution with amines including a number of L- and D-amino acids and hydrazine. The afforded protein hydrazides could be used further for expressed protein ligation. We demonstrated the versatility of this approach with the successful synthesis of ubiquitin conjugates, ubiquitin-like protein conjugates, histone H2A with a posttranslational modification, RNAse H that actively hydrolyzed RNA, and exenatide that is a commercial therapeutic peptide. The technique, which is exceedingly simple but highly useful, expands to a great extent the synthetic capacity of protein chemistry and will therefore make a large avenue of new research possible.

Site-specific labeling of DNA–protein conjugates by means of expressed protein ligation

2007 ◽  
pp. 353-355 ◽  
Author(s):  
Marina Lovrinovic ◽  
Ljiljana Fruk ◽  
Hendrik Schröder ◽  
Christof M. Niemeyer
Keyword(s):  
Expressed Protein Ligation ◽  
Site Specific ◽  
Protein Ligation ◽  
Protein Conjugates

scholarly journals Analysis of Protein Kinase Autophosphorylation Using Expressed Protein Ligation and Computational Modeling

2008 ◽  
Vol 130 (17) ◽  
pp. 5667-5669 ◽  
Author(s):  
Kerry A. Pickin ◽  
Sidhartha Chaudhury ◽  
Blair C. R. Dancy ◽  
Jeffrey J. Gray ◽  
Philip A. Cole
Keyword(s):  
Protein Kinase ◽  
Computational Modeling ◽  
Expressed Protein Ligation ◽  
Protein Ligation

Expressed Protein Ligation

2008 ◽  
pp. 537-566 ◽  
Author(s):  
Matthew R. Pratt ◽  
Tom W. Muir
Keyword(s):  
Expressed Protein Ligation ◽  
Protein Ligation

scholarly journals The Yeast ULP2 (SMT4) Gene Encodes a Novel Protease Specific for the Ubiquitin-Like Smt3 Protein

2000 ◽  
Vol 20 (7) ◽  
pp. 2367-2377 ◽  
Author(s):  
Shyr-Jiann Li ◽  
Mark Hochstrasser
Keyword(s):  
Cell Function ◽  
Feedback Mechanism ◽  
Cell Cycle Checkpoint ◽  
Temperature Sensitive ◽  
Partial Recovery ◽  
Protein Ligation ◽  
Protein Conjugates ◽  
Dna Damaging Agents ◽  
Cycle Checkpoint ◽  
Specific Protease

ABSTRACT Yeast Smt3 and its vertebrate homolog SUMO-1 are ubiquitin-like proteins (Ubls) that are reversibly ligated to other proteins. LikeSMT3, SMT4 was first isolated as a high-copy-number suppressor of a defective centromere-binding protein. We show here that SMT4 encodes an Smt3-deconjugating enzyme, Ulp2. In cells lacking Ulp2, specific Smt3-protein conjugates accumulate, and the conjugate pattern is distinct from that observed in a ulp1ts strain, which is defective for a distantly related Smt3-specific protease, Ulp1. The ulp2Δ mutant exhibits a pleiotropic phenotype that includes temperature-sensitive growth, abnormal cell morphology, decreased plasmid and chromosome stability, and a severe sporulation defect. The mutant is also hypersensitive to DNA-damaging agents, hydroxyurea, and benomyl. Although cell cycle checkpoint arrest in response to DNA damage, replication inhibition, or spindle defects occurs with normal kinetics, recovery from arrest is impaired. Surprisingly, either introduction of aulp1ts mutation or overproduction of catalytically inactive Ulp1 can substantially overcome theulp2Δ defects. Inactivation of Ulp2 also suppresses several ulp1ts defects, and the double mutant accumulates far fewer Smt3-protein conjugates than either single mutant. Our data suggest the existence of a feedback mechanism that limits Smt3-protein ligation when Smt3 deconjugation by both Ulp1 and Ulp2 is compromised, allowing a partial recovery of cell function.

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