Advantages: Direct stabilization and co-translational integration of membrane proteins into nanodiscs; useful for toxic proteins
Amount of purified protein: Highest among our expression systems
SYSTEM 2
BACULOVIRUS INSECT SYSTEM
Advantages: Near mammalian-like post-translational modifications; Easy to handle eucaryotic expression system
Amount of purified protein: Higher than mammalian cell lines
SYSTEM 3
HEK293 CELLS
Advantages: Homolog mammalian protein expression
Amount of purified protein: Lowest of the three, but with the highest authenticity among our systems
In contrast to many E. coli-based systems, we mainly focus on eukaryotic expression systems. This is especially relevant for the many human proteins of our off-the-shelve projects, as these systems allow us to produce the proteins with all important post-translational modifications. Especially from a medical point of view, this makes sense because it brings us as close as possible to the in vivo conditions of the human body.
We typically start with sequence-optimized, full-length genes expressing the wild type protein. In accordance with the project's requirements, different constructs with different affinity tags and positions can be included.
The optimization of protein expression is carried out to the full extent. Next to the expression systems, features that we optimize for maximum protein yield are:
Promoter regions (strong/weak expression, tightness of control)
Type and position of the affinity purification tag
Optional: best-expressing domains, including a boundary screen of domain borders
After discovering the ideal expression conditions for your protein of interest, we can scale up the expression. This way we can produce larger amounts of your protein in a shorter time period.
Protein Solubilization
Protein Solubilization
Essential step to obtain structurally intact and functional membrane proteins. The core part consists of our NativeMPTM Technology Platform.
NativeMPTM Platform:
Solve any structure for compound verification
Produce in mg quantities for assay development and studies
Facilitate lead optimization
Perform assays at body temperature
Biotinylation for immobilization possible
Copolymer Nanodiscs: Copolymers like AASTY, DIBMA, SMA, UltrasoluteTM Amphipol, and our very own Cubipol can form complexes that are called copolymer nanodiscs. During the last decades, they have increasingly started to replace detergents as the most popular tool for membrane protein solubilization. Their two key advantages: They combine the essential steps of membrane protein solubilization and subsequent stabilization into one step, and they retain the native host cell lipid environment.
Application Case Study (GLP1R)
NativeMPTM Platform enables stabilization of type-2 Diabetes target GLP1R.
➡ Explore Application Data
Protein Stabilization
Protein Stabilization
Traditionally seen as a separate step to solubilization, the introduction of copolymers has merged these two steps. For older, non-native methods such as MSP nanodiscs or even detergents, stabilization is of course still an independent, additional step. Its goal is to stabilize membrane proteins and keep them functional for ensuing assays and applications.
Utilizing our NativeMPTM Platform, we can highly adapt the selection of copolymers based on the customers downstream requirements and needs. Constant further development of our technologies, most recently with the new Cubipol, allows us to always offer our customers the best possible approach, far ahead of mass production.
While copolymers are currently the most advanced technology, we still offer our customers the option of requesting older technologies such as MSP nanodiscs or detergents for projects.
Copolymer Nanodiscs: As an innovative advancement, copolymers have the capacity to solubilize and stabilize a membrane protein. The protein remains surrounded by its natural lipid composition. These copolymer-lipid complexes have never seen detergents at all. Utilizing copolymer nanodiscs will open new ways of research and deeper insights into protein functionalities! You can find more information about this technology on our copolymer wiki page.
Protein Purification
Protein Purification
After the membrane protein has been stabilized or a soluble protein has been expressed, it is time to purify the said protein of interest. To extract the desired protein from the rest of the cell's components after cell lysis, Cube Biotech offers numerous applicable options here:
Surface affinities/affinity tags: Our favorite affinity tag for membrane protein purification is the Rho1D4 tag. Since it is an antibody-based affinity tag it provides incredible specificity and high yields. We are, however, open to discussing and using other affinity tags in your project if you like. Since we are also manufacturers of matching purification products, we can ensure that we only work with the best-suited products for your protein purification assays. See this page for more information about the affinity tags that we usually work with and produce matching purification products for.
Customized agarose resins/magnetic beads: On request, we produce a specialized protein purification matrix just for you. It can include a protein-specific antibody, a natural ligand of the protein of interest, or other components specialized for your needs. Click here to discover more about the custom resin that we have already created on request.
Protein Characterization
Protein Characterization
All previous efforts aim to gain a protein sample for this step, to verify its quality and activity. Now it is time to identify the characteristics of your protein of interest. For this purpose we offer:
DLS and MST Characterization Data (GLP1R, EGFR, TLR5)
NativeMPTM Platform enables characterization of three highly relevant targets GLP1R, EGFR, and TLR5.
➡ Explore Characterization Data
ELISA
ELISA - a short introduction
ELISA (Enzyme-Linked Immunosorbent Assay) is widely used in biotechnology for the detection and quantification of specific proteins or antigens. Its high sensitivity and specificity make it valuable for diagnosing diseases, studying immune responses, and assessing biomarker levels in clinical samples. Additionally, it is commonly used in drug discovery to monitor protein-protein or antibody-antigen interactions.
Dynamic Light Scattering (DLS)
What is DLS used for?
Dynamic Light Scattering (DLS) is a technique used to determine the size and distribution of particles in solution, including proteins and nanoparticles. In biotechnology, it is crucial for analyzing protein aggregation, studying drug delivery systems, and evaluating the stability of biological formulations. DLS offers a non-invasive and fast method for monitoring the hydrodynamic radius and polydispersity of macromolecules in solution.
Protein Stability Assays
What are protein stability assays?
Protein stability assays are essential for assessing the thermal and chemical stability of proteins, which is critical in drug development and formulation. Techniques like differential scanning fluorimetry (DSF) and thermofluor assays help identify the conditions under which proteins denature or aggregate. These assays are valuable for optimizing storage conditions and ensuring the longevity and efficacy of therapeutic proteins.
Surface Plasmon Resonance (SPR)
Where does SPR play a role?
Surface Plasmon Resonance (SPR) allows real-time, label-free analysis of biomolecular interactions, making it a key tool in drug discovery and biosensor development. SPR provides kinetic and affinity data on interactions between proteins, antibodies, or small molecules with high precision. This technology is particularly useful for characterizing binding events, understanding molecular mechanisms, and screening potential drug candidates.
MST
MST explained
Microscale Thermophoresis (MST) measures molecular interactions by detecting changes in the mobility of molecules in a temperature gradient. MST can quantify binding affinities and study interactions between proteins, nucleic acids, and small molecules under native conditions. Its versatility and low sample consumption make it a valuable tool in biophysics and drug discovery research.
Protein Structure Determination
Protein Structure Determination
One of the key characteristics of a protein is its 3D structure. Unfortunately, it is also one of the hardest characteristics to identify. At the moment Cryo-EM is the leading method for protein structure determination. For membrane proteins, Cryo-EM is the most plausible option to identify their tertiary structure as other methods fall short here and may lead to unsatisfying results. Especially copolymers can be highlighted here as a valuable technology for the structural determination of membrane proteins.
One of our next-gen Cubipol copolymers, allowed us to obtain the previously unsolvable structure of the 'human' P2X4 ion channel in its native membrane environment. P2X4, a crucial player in synaptic transmission and inflammation, holds significant promise in drug discovery, particularly for developing novel therapies for chronic pain and inflammatory diseases. Traditionally, detergents have been the standard method for stabilizing membrane proteins for cryo-EM. However, they fall short of preserving the protein’s native environment, affecting functional stability and further analysis.
Cubic Phase Crystallization
The classical way to solve 3D structures of membrane proteins. Cubic Phase crystallization has already solved countless tertiary structures of membrane proteins. Our company is the patent owner of the CIMP (controlled in-meso phase crystallization) method which combines the lipid cubic phase and vapor diffusion. Therefore we have gathered lots of experience with this method. Fun fact: Our Company was named after the Cubic Phase Crystallization method.
Other Customized Services
Proteogenix
Besides our standard methods of surface affinity tags or customized agaroses and resins, together with our partners of Proteogenix we are also able to produce target specific antibodies with our NativeMPTM platform for you.
Momentum Biotechnologies
This partnership opens new ways for mass spectrometry-based drug discovery. Leverage novel high-throughput approaches for interrogating highly clinically relevant membrane-bound proteins.
Soluble Proteins
Soluble Proteins
Although purification and stabilization of membrane proteins is Cube Biotech's specialty, it should also be clear at this point that we can arrange protein services for soluble proteins as well. Projects involving DNA-binding proteins, such as transcription factors or the purification of lipid-associated proteins, can be requested through us at any time.
