For Research Use Only. Not for Clinical Use.

Creative Biolabs has a strong biotechnology platform, with customers and partners all over the world. We have established a half-life assay platform covering a variety of services. Half-life Assay by the Promoter-reference technique (PRT) is one of the quality-guaranteed services we provide.

The Promoter-reference Technique

One of the biochemical techniques that have been developed recently to monitor protein degradation in eukaryotic system is the PRT. This method has been exploited to identify the Pro-N-end rule pathway. The N-end rule pathway is a set of proteolytic systems whose unifying feature is their ability to recognize and polyubiquitylate proteins containing N-terminal degradation signals called N-degrons, thereby causing the degradation of these proteins by the proteasome. Most proteins in a cell are conditionally short-lived N-end rule substrates, either as full-length proteins or as protease-generated natural protein fragments. Using the PRT, the ratio between levels of a test protein fragment to a long-lived stable reference protein fragment can be measured. Epitope-tagged reference and test proteins were coexpressed from two identical promoters containing additional DNA elements developed by others. Once transcribed into an mRNA, these elements form 5'-RNA aptamers that can bind to gene-specific translation inhibitors, thereby repressing translation in cis.

Fig.1 The Promoter-reference Technique (Eldeeb, 2019)

Key Features of PRT

• Advantages:
1. A coexpression, from identical transcriptional promoters, of a test protein and a long-lived reference protein.
2. A gene-specific inhibition of translation during a chase, which is extremely important in avoiding a global shut off of translation by global protein translation inhibitors such as Cycloheximide. This aspect is particularly crucial if any of the relevant components of the degradation machinery is itself short-lived.
• Disadvantages:
1. The use of labeling or fusion technology in the context of heterologous proteins is not yet mature.
2. The nature of the tag and/or the use of this fusion may potentially impact the degradation rate or the recognition of target protein fragments by the degradation machinery. Nevertheless, all these limitations are common for protein fused with tags, which are widely used in molecular biological research currently.

Nevertheless, all these limitations are common for protein fused with tags, which are widely used in molecular biological research currently.

Reference

1. Eldeeb, M. A.; et al. A molecular toolbox for studying protein degradation in mammalian cells. Journal of neurochemistry. 2019,151(4): 520-533.