Protein Services
As a team of passionate (membrane) protein scientists, we offer small to mid-scale, non-GMP protein services for a broad range of applications. Individual work packages made up of our modular structure can be tailored to your specific needs. View our introduction video to our services to learn more.
The modular structure of our Protein Services
| Why Cube Biotech's Protein Services? | |||
|---|---|---|---|
| There are numerous reasons to choose Cube Biotech. Here are a few. | |||
 Versatile skills More than 400 proteins were successfully expressed, stabilized, and purified! |  Time efficient E.g. 30 mg wild type membrane protein stabilized in nanodisc produced in 10 weeks! |  Success where others fail! 95 % success rate for membrane protein expression and stabilization! |  Internationally approved! Over 30 projects with „Big Pharma“ completed! |
Module 1: Protein Expression
As a first step, we determine the right expression system for your protein. Besides E.coli, we also use the Baculovirus expression system in Sf9 or T. ni (High FiveTM) insect cells (up to 50 L scale). Furthermore, we use transient transfection in HEK 293 suspension cells (up to 30 L scale) and our E. coli-based cell-free expression system in combination with different stabilization systems.
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.
We are optimizing the expression conditions to the fullest. Next to the expression systems, features that we optimize for maximum protein yield are:
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.
| Expression system | Advantages | Amount of purified protein | Illustration |
|---|---|---|---|
| Cell-free expression system: |
| Highest among our expression systems. |  |
| Baculovirus Insect system |
| Higher than mammalian cell lines |  |
| Hek293 cells | Homolog mammalian protein expression | Lowest of the three, but with the highest authenticity among our systems |  |
We are optimizing the expression conditions to the fullest. Next to the expression systems, features that we optimize for maximum protein yield are:
- Expression conditions (media, temperature, induction conditions)
- 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.
Module 2: Protein solubilization
This step is essential to obtain structural intact and functional membrane proteins. Our membrane protein service contains two different methods for this step that you can choose from:
Ultrapure Detergents: Detergents are the classic approach to solubilize membrane proteins. Using them requires a screening process to identify the detergents that fit your membrane protein best. Of course, Cube Biotech will perform this screening process. Detergent screening parameters are:
Ultrapure Detergents: Detergents are the classic approach to solubilize membrane proteins. Using them requires a screening process to identify the detergents that fit your membrane protein best. Of course, Cube Biotech will perform this screening process. Detergent screening parameters are:
- Detergent Concentration
- Solubilization time
- Buffer composition
Synthetic Polymers: Polymers like SMA and DIBMA can form complexes that are called synthetic nanodiscs. During the last decades, they have increasingly started to replace detergents as the most popular tool for membrane protein solubilization. Their key advantage: They combine the essential steps of membrane protein solubilization and subsequent stabilization. This basically fuses our modules 2 and 3.
Module 3: Protein Stabilization
This module is the next step after protein solubilization. Its goal is to stabilize membrane proteins and keep them functional after their native cell membrane has been removed. The main idea is to mimic the original cell membrane of the membrane protein of interest to ensure maximal authenticity with its functions. Cube Biotech offers three options to archive membrane protein stabilization.
1. Detergents: The traditional way to stabilize membrane proteins. We offer a great choice of different detergents. Of course, we will help you screen for the detergent that best suits your downstream application.
Figure 2: Membrane protein (orange) stabilized through detergents (black).2. MSP Nanodiscs: This method has several advantages over traditional detergent-based approaches for membrane protein stabilization. With their controlled phospholipid composition, a near-perfect replication of the native membrane environment of the membrane protein can be created. The handling of nanodiscs, in general, is one of Cube Biotech's strongest assets. Years of unmatched experience make Cube Biotech the best option for MSP nanodisc-related projects.
Figure 3: A membrane protein stabilized inside an MSP nanodisc.3. Synthetic Nanodiscs: As an innovative way, the synthetic polymer DIBMA has the capability to solubilize and stabilize a membrane protein. Interestingly the protein remains surrounded by its natural lipid composition in a nanodisc. These complexes have never seen detergents at all. Using Synthetic polymers like DIBMA leaves the original membrane environment of the membrane protein of interest intact! More information about DIBMA.
Figure 4: Membrane protein (orange) stabilized inside a synthetic nanodisc. Its native phospholipids (grey) were cut out together with the protein out of the original cell membrane by the synthetic polymer DIBMA (blue).Module 4: Protein Purification
After the membrane protein has been stabilized in module 3 or a soluble protein has been expressed in module 1 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 options that can be applied here.
1. 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.
Figure 5: Cube Biotech's PureCube INDIGO-NI resin inside a column ready for protein purification.2. 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 on this link to discover more about the custom resin that we have already created on request.
Figure 6: Custom Resins use a 100% specific affinity towards your protein of interest.Module 5: Protein Characterization
All previous modules 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 do offer:
- Dynamic light scattering (DLS)
- Surface Plasmon resonance (SPR)
- ELISA
- Thermostability
- Protein Stability Assays
Figure 7: Our experts on the job for your project!Module 6: 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. Cube Biotech offers two types of structure determination methods for your membrane protein.
Cryo-EM sample preparation: At the moment Cryo-EM is the state-of-the-art 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.
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.
Figure 8: Cryo-EM has one of the best resolutions for protein structures to date!Watch our Cryo-EM Video
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.
In case you want to crystallize your membrane protein in-house we offer our MO plates for Cubic phase crystallization.
Fun fact: Our Company was named after the Cubic Phase Crystallization method.
In case you want to crystallize your membrane protein in-house we offer our MO plates for Cubic phase crystallization.
Figure 9: Protein crystal in the cubic phaseOur service introduction video - learn why we are your best option for membrane protein-related projects.
Interested in our protein services? Fill out the form below to sent your request!
Contact
Viewed