Synthesis and purification of linkage-specific polyubiquitin chains of distinct length for structural studies

Anshumali Mittal; Binita Shakya

doi:10.1016/j.ab.2018.08.007

Synthesis and purification of linkage-specific polyubiquitin chains of distinct length for structural studies

Anal Biochem. 2018 Oct 15:559:1-4. doi: 10.1016/j.ab.2018.08.007. Epub 2018 Aug 11.

Authors

Anshumali Mittal¹, Binita Shakya²

Affiliations

¹ Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, USA. Electronic address: Anshumali.Mittal@tufts.edu.
² Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, USA.

PMID: 30107157
DOI: 10.1016/j.ab.2018.08.007

Abstract

Polyubiquitylation is one of the most versatile post-translational modifications involved in the regulation of numerous intracellular signaling processes. An assembly procedure that is simple, robust, and efficient to synthesize and purify linkage-specific polyubiquitin chains of defined length at a preparative scale is required in biophysical and structural studies. Here, we have optimized known enzymatic procedures in the form of a protocol to obtain multi-milligrams of Lys48-and Lys63-linked polyubiquitin chain types with more than 99% purity. Mass spectrometry (ESI/MS) analysis of K48- and K63-linked diubiquitin confirmed that the enzymes used in the preparation generated homogeneous linkages with no promiscuity.

Keywords: Chromatography; K48- and K63 linked polyubiquitin chains; Ubiqutin.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Mass Spectrometry
Polyubiquitin / chemical synthesis*
Polyubiquitin / chemistry
Polyubiquitin / isolation & purification*
Protein Conformation

Substances

Polyubiquitin