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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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Related Experiment Video

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Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins
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Analyzing protein posttranslational modifications using enzyme-catalyzed expressed protein ligation.

Niyi Adelakun1, Jordan Parrish1, Nam Chu1

  • 1Department of Cancer Biology and Genetics, the Comprehensive Cancer Center, College of Medicine, The Ohio State University, Columbus, OH, United States.

Methods in Enzymology
|March 22, 2023
PubMed
Summary

Enzyme-catalyzed expressed protein ligation (EPL) enables Cysteine-free peptide ligation, expanding protein modification capabilities. This method facilitates site-specific incorporation of multiple posttranslational modifications (PTMs) for biochemical studies.

Keywords:
EnzymologyPeptide synthesisProtein posttranslational modificationsProtein semisynthesisSubtiligase

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Chemistry

Background:

  • Expressed protein ligation (EPL) is a method for site-specific protein modification.
  • Traditional EPL requires a Cysteine residue at the ligation site, limiting its application.
  • Posttranslational modifications (PTMs) are crucial for protein function and are often studied using modified proteins.

Purpose of the Study:

  • To develop a novel method for expressed protein ligation that does not require a Cysteine residue.
  • To expand the utility of EPL for incorporating complex modifications, such as multiple PTMs.
  • To demonstrate the applicability of this new method for studying protein function.

Main Methods:

  • Enzyme-catalyzed expressed protein ligation (EPL) using subtiligase.
  • Generation of protein C-terminal thioesters and Cysteine-free peptides.
  • Purification of the ligated protein product.

Main Results:

  • Successfully ligated Cysteine-free peptides to protein thioesters using subtiligase.
  • Demonstrated the incorporation of site-specific phosphorylations onto the C-terminal tail of PTEN.
  • Obtained substantial yields of the modified protein for further analysis.

Conclusions:

  • Enzyme-catalyzed EPL provides a versatile alternative to traditional EPL, overcoming the Cysteine limitation.
  • This method enables the efficient generation of site-specifically modified proteins with complex PTMs.
  • The developed technique is valuable for biochemical and biophysical studies of protein function and regulation.