For Research Use Only. Not for Clinical Use.

Creative Biolabs is well-known and competitive in the field of drug half-life. Based on our advanced platforms and extensive experience, we can provide half-life services such as half-life assay by the Ubiquitin-reference technique for our clients all over the world.

Introduction of the Ubiquitin-Reference Technique

The degradation of proteins in eukaryotic cells mainly occurs through two pathways: autophagy and the ubiquitin-proteasome system. In the process of ubiquitin-proteasome mediated protein degradation, a series of ubiquitinase cascade reactions mark the substrate protein with ubiquitin so that it can be recognized and degraded by the proteasome. Researchers found that the half-life of a protein depends on the type and characteristics of its N-terminal amino acid residues, which is called the N-end rule. The corresponding degron is dubbed N-degron. The underlying pathway (the N-end rule pathway) is a crucial part of the ubiquitin-proteasome system in eukaryotes. Notably, despite the absence of ubiquitin from prokaryotes, they do have versions of N-end rule pathways.

Since the discovery of the N-end rule in 1986, several methods exploited the use of Ubiquitin (Ub) fusions in the design of some expression plasmids to study and monitor the rate of proteolysis of some protein fragments in the context of N-end rule in prokaryotic and eukaryotic systems. One of these toolkits was the ubiquitin-reference technique (URT), which addressed the issue of scattering data in the conventional pulse-chase degradation assays.

Fig.1 Ubiquitin reference technique and bacterial N-end rule pathways. (Piatkov, 2013)

The Principle of URT

The URT approach relies on the design of a fusion in which the Ub moiety is positioned in the middle between an upstream polypeptide (a metabolically stable reference protein fragment) and a downstream polypeptide (the test protein fragment). Upon expressing this construct in cells, ubiquitin hydrolases mediate the co-translational cleavage of a URT fusion after the last residue of Ub to generate a test protein fragment with a desired N-terminal amino acid residue and a reference protein that encompasses the C-terminal Ub moiety. One can determine the relative levels of the test protein fragment against the long-lived reference protein in the same sample, if both the reference protein fragment and the test protein fragment are immunoprecipitated and subsequently analyzed in a pulse-chase analysis procedure.

The Application of URT

The URT assay may allow the compensation for the variation of data with respect to labeling efficiency, immunoprecipitation yields, sample volumes, and other sources of sample-to-sample variation. In addition to being more accurate than pulse-chases without a reference, URT permits the possibility of detection and monitoring of the degradation of a test protein during the pulse phase (before the chase phase). The latter feature of URT permits the detection and the subsequent monitoring of the degradation of a protein that occurs either co-translationally or shortly after its completion.

Reference

1. Piatkov, K.; et al. Ubiquitin reference technique and its use in ubiquitin-lacking prokaryotes. PloS one. 2013, 8(6), e67952.