Our Technology

 

The Ubiquitin Proteasome System (Fig 1) (UPS) is a highly regulated pathway which controls the principal functions of almost all the cellular proteins in both the nucleus and the cytosol of eukaryotic cells. The UPS not only plays a vital role in protein degradation, but also acts as a key regulator of a wide variety of cellular processes, such as chromatin structure and function, protein trafficking, autophagy, and mitophagy. Because of the wide-reaching impact of ubiquitin and the UPS, failures in this system often contribute directly or indirectly to the pathogenesis of many diseases, including cancer, inflammation, and neurodegeneration.

At LifeSensors, our technology is focused on the ubiquitin pathway and we have developed highly innovative reagents, assays, and platforms to assist you in your research and drug discovery efforts.

 

Ubiquitin and Ubiquitin Chains

Ubiquitin is a small polypeptide that is conjugated via its C-terminus to an ε-amino group of lysine on a target protein; this conjugation is referred to as monoubiquitylation. Subsequently, additional ubiquitin moieties can be conjugated to the initial ubiquitin, utilizing any of seven lysine residues (K6, K11, K27, K29, K33, K48, K63) on the surface of ubiquitin; this ubiquitin chain formation is referred to as polyubiquitylation. Enzymatic conjugation of ubiquitin is performed by a series of enzymes — ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2, and ubiquitin ligase E3.  Ubiquitylation of proteins is reversible in cells; both mono- and polyubiquitylated chains are cleaved by hydrolysis catalyzed by deubiquitylases (DUBs).

LifeSensors has considerable experience with enzymes at all levels of the ubiquitin pathway, including E1, E2, and E3 enzymes and DUBs. LifeSensors’ extensive knowledge of protein purification has enabled us to produce approximately 30 E2 enzymes, 20 E3 enzymes, and 35 DUBs, with the capability for custom expression available. Further, our extensive panel of assays simplifies the process of confirming enzyme activity and determining the compatibility of E2-E3 pairs and ensures that you can choose an enzyme that will best address specific, critical biological questions that you wish to explore.

LifeSensors has developed a variety of ubiquitin derivatives for your research, including DUB-resistant polyubiquitin chains for binding interaction studies, ubiquitin conjugates for DUB assays and screening (fluorophores and reporter enzymes), selective ubiquitin chains (di-, tri-, and tetra-ubiquitin chains) for DUB specificity and binding assays, and site-specific lysine ubiquitin mutants for E3 ligase and DUB selectivity assays.

 

Tandem Ubiquitin Binding Entities (TUBEs)

Traditional strategies for characterizing ubiquitylated proteins often require immunoprecipitation of overexpressed epitope-tagged ubiquitin or the use of ubiquitin antibodies. Antibody based ubiquitin proteomics is expensive for large scale studies, and, moreover, ubiquitin antibodies sold by most suppliers are notoriously non-selective, leading to artifacts. LifeSensors has developed technology to specifically isolate polyubiquitylated proteins from cell lysates and tissues using Tandem Ubiquitin Binding Entities (TUBEs).

TUBEs are tandem ubiquitin binding domains (UBDs) that bind tetra-ubiquitin with Kds in the nanomolar range. TUBEs have also been demonstrated to protect ubiquitylated proteins from both deubiquitylation and proteasome-mediated degradation, even in the absence of inhibitors normally required to block such activities.

Our technology has transformed the ubiquitin community due to the powerful ability to selectively capture polyubiquitylated proteins. In addition to development of TUBEs that bind to all types of polyubiquitin chains (pan-TUBEs), LifeSensors has developed chain selective  TUBEs that bind selectively to K48 or K63, or linear (M1) polyubiquitylated proteins. The latter are sold as K48 HF TUBEs, K63 TUBEs and M1 TUBEs, respectively. Utilizing pan-selective and chain-selective TUBEs with nanomolar affinity, LifeSensors has developed a variety of assays and services utilizing TUBE technology.

Our assays include:

  • E3 ELISA Assays (FIG 2) – high-throughput system for monitoring E3 ligase activity
  • TR-FRET E3 Assays (FIG 3) – fluorescence-based high-throughput assay system for screening compound libraries against E3 ligase activity
  • UbiTest (FIG 4) – a TUBE-based pull-down method that isolates total cellular ubiquitylated proteins. Subsequently, samples are treated with pan-selective DUBs to remove polyubiquitin chains. The target protein is identified by its native molecular weight and analyzed and quantified by immunoblotting. UbiTest is one of the most sensitive methods available to quantify ubiquitylation levels of proteins in vivo.
  • UbiQuant (FIG 5) – the most sensitive and accurate method for determining the concentration of total ubiquitin (poly + mono) in cell and tissue lysates as a means of monitoring the effects of compounds such as PROTACs, E3 ligase inhibitors, DUB inhibitors, and proteasome inhibitors on cellular proteins.
  • UbiQuant S ELISA (FIG 6) and AlphaLISA (FIG 7) – enable accurate determination of substrate ubiquitylation for monitoring the effects of various treatments on patterns of cellular substrate ubiquitylation.
  • UbiQuant Ultra –the latest member of the UbiQuant family of assays, it entails plates coated with high affinity TUBEs, maximizing the likelihood that the ubiquitylated portion of the POI will be captured and quantified.

 

Our services utilizing TUBE technology include ubiquitin E3 ligase screening and profiling, TUBE-based mass spec ubiquitin proteomics, PROTAC drug discovery, and biomarker assay development.

 

Difficult to Express Proteins and SUMO Technology

Production of recombinant proteins routinely encounters problems, including formation of inclusion bodies, incorrect protein conformation, toxicity to the host cell, and low protein yield. These issues are most often addressed by using an alternative host or genetically fusing the protein of interest (POI) to a carrier protein (fusion tag).  Attachment of the C-terminus of SUMO to the N-terminus of a protein of interest  can dramatically improve protein solubility, achieve native protein folding, and increase total yield by improving expression and decreasing degradation. LifeSensors has pioneered SUMO (Small Ubiquitin Like Modifier) fusion technology, which increases the quantity and quality of numerous recombinant proteins in both prokaryotic and eukaryotic expression hosts.

Our SUMO-tag expression systems (FIG 8) maximize the yield of soluble, functional proteins in E. coli. SUMO functions as both a chaperonin and initiator of protein folding, improving the solubility and yield of your POI. De-sumoylases remove the SUMO tags, releasing your protein with the desired N-terminal amino acid. Both SUMO tags and de-sumoylases contain 6xHis tags allowing efficient subsequent removal, leaving pure protein.

The SUMO Pro system is designed for expression and purification of proteins from E. coli. Since all eukaryotic cells contain SUMO and SUMO proteases, the latter of which will cleave SUMO-fusion proteins immediately following expression, LifeSensors has engineered SUMOstar, a SUMO tag for use in eukaryotic hosts, which is resistant to endogenous proteases, thus preserving enhanced expression, increased solubility and native folding.  SUMOstar fusion proteins can be cleaved only with SUMOstar protease.