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:

Amer, Brendan R. ^[1]; Macdonald, Ramsay ^[1]; Jacobitz, Alex W. ^[1]; Liauw, Brandon ^[1]; Clubb, Robert T. ^[2]

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+ Show Author Affiliations

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:

Sat Feb 06 00:00:00 EST 2016

Research Org.:

Univ. of California, Los Angeles, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

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