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Title: Rapid addition of unlabeled silent solubility tags to proteins using a new substrate-fused sortase reagent

Abstract

We report that many proteins can’t be studied using solution NMR methods because they have limited solubility. To overcome this problem, recalcitrant proteins can be fused to a more soluble protein that functions as a solubility tag. However, signals arising from the solubility tag hinder data analysis because they increase spectral complexity. We report a new method to rapidly and efficiently add a non-isotopically labeled Small Ubiquitin-like Modifier protein (SUMO) solubility tag to an isotopically labeled protein. The method makes use of a newly developed SUMO-Sortase tagging reagent in which SUMO and the Sortase A (SrtA) enzyme are present within the same polypeptide. The SUMO-Sortase reagent rapidly attaches SUMO to any protein that contains the sequence LPXTG at its C-terminus. It modifies proteins at least 15-times faster than previously described approaches, and does not require active dialysis or centrifugation during the reaction to increase product yields. In addition, silently tagged proteins are readily purified using the well-established SUMO expression and purification system. The utility of the SUMO-Sortase tagging reagent is demonstrated using PhoP and green fluorescent proteins, which are ~90 % modified with SUMO at room temperature within four hours. SrtA is widely used as a tool to construct bioconjugates.more » Lastly, significant rate enhancements in these procedures may also be achieved by fusing the sortase enzyme to its nucleophile substrate.« less

Authors:
 [1];  [1];  [1];  [1];  [2]
  1. Univ. of California, Los Angeles, CA (United States). Department of Chemistry and Biochemistry and UCLA-DOE Institute of Genomics and Proteomics
  2. Univ. of California, Los Angeles, CA (United States). Department of Chemistry, Biochemistry and UCLA-DOE Institute of Genomics and Proteomics and Molecular Biology Institute
Publication Date:
Research Org.:
Univ. of California, Los Angeles, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1466770
Grant/Contract Number:  
FC02-02ER63421
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biomolecular NMR
Additional Journal Information:
Journal Volume: 64; Journal Issue: 3; Journal ID: ISSN 0925-2738
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Silent solubility tag; Sortase; Protein ligation; SUMO

Citation Formats

Amer, Brendan R., Macdonald, Ramsay, Jacobitz, Alex W., Liauw, Brandon, and Clubb, Robert T. Rapid addition of unlabeled silent solubility tags to proteins using a new substrate-fused sortase reagent. United States: N. p., 2016. Web. doi:10.1007/s10858-016-0019-z.
Amer, Brendan R., Macdonald, Ramsay, Jacobitz, Alex W., Liauw, Brandon, & Clubb, Robert T. Rapid addition of unlabeled silent solubility tags to proteins using a new substrate-fused sortase reagent. United States. doi:10.1007/s10858-016-0019-z.
Amer, Brendan R., Macdonald, Ramsay, Jacobitz, Alex W., Liauw, Brandon, and Clubb, Robert T. Sat . "Rapid addition of unlabeled silent solubility tags to proteins using a new substrate-fused sortase reagent". United States. doi:10.1007/s10858-016-0019-z. https://www.osti.gov/servlets/purl/1466770.
@article{osti_1466770,
title = {Rapid addition of unlabeled silent solubility tags to proteins using a new substrate-fused sortase reagent},
author = {Amer, Brendan R. and Macdonald, Ramsay and Jacobitz, Alex W. and Liauw, Brandon and Clubb, Robert T.},
abstractNote = {We report that many proteins can’t be studied using solution NMR methods because they have limited solubility. To overcome this problem, recalcitrant proteins can be fused to a more soluble protein that functions as a solubility tag. However, signals arising from the solubility tag hinder data analysis because they increase spectral complexity. We report a new method to rapidly and efficiently add a non-isotopically labeled Small Ubiquitin-like Modifier protein (SUMO) solubility tag to an isotopically labeled protein. The method makes use of a newly developed SUMO-Sortase tagging reagent in which SUMO and the Sortase A (SrtA) enzyme are present within the same polypeptide. The SUMO-Sortase reagent rapidly attaches SUMO to any protein that contains the sequence LPXTG at its C-terminus. It modifies proteins at least 15-times faster than previously described approaches, and does not require active dialysis or centrifugation during the reaction to increase product yields. In addition, silently tagged proteins are readily purified using the well-established SUMO expression and purification system. The utility of the SUMO-Sortase tagging reagent is demonstrated using PhoP and green fluorescent proteins, which are ~90 % modified with SUMO at room temperature within four hours. SrtA is widely used as a tool to construct bioconjugates. Lastly, significant rate enhancements in these procedures may also be achieved by fusing the sortase enzyme to its nucleophile substrate.},
doi = {10.1007/s10858-016-0019-z},
journal = {Journal of Biomolecular NMR},
number = 3,
volume = 64,
place = {United States},
year = {2016},
month = {2}
}

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