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 from 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 Serives
| Why Cube Biotech's Protein Services? | |||
|---|---|---|---|
| There are numerous reasons to choose Cube Biotech. Here are a few. | |||
 Versatile skills More than 250 proteins 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 transcient 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 to the requirements of the project 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 | Homologue mammalian protein expression | Lowest of the three, but with 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 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 strucutral 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 fits 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 fits your membrane protein best. Of course Cube Biotech will perform this screening process. Detergent screening parameters are:
- Detergent Concentration
- Solubilization time
- Buffer composition
Synthetic Polyers: 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 proteins 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 keeping 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 enviroment of the membrane protein can be created. The handling of nanodiscs in general is one of Cube Biotech's strongest assests. Years of unmatched experience are making Cube Biotech the best option for MSP nanodisc related projects.
Figure 3: A membrane protein stabilized inside a 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 leave the original membrane enviroment of the membrane protein of interest intact! More information about DIBMA.
Figure 4: Membrane protein (orange) stabilized inside a synthetic nanodiscs. 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 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 favourite affinity tag for membrane protein purification is the Rho1D4 tag. Since it is antibody based affinity tag it provides incredible specificity and high yields. We are open to discuss and use other affinity tags in your project if you like to. Since we are also manufacturers of the 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 purificaiton.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: Overview of custom resins that we have already created on request.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: Overview of custom resins that we have already created on request.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 patent owner of the CIMP (controlled in-meso phase crystallization) method (controlled in-meso phase crystallization) which combines the lipid cubic phase and vapor deffusion. 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 inhouse 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 inhouse we offer our MO plates for Cubic phase crystallization.
Figure 9: Protein crystal in cubic phaseOur service introduction video - learn why we are your best option for membrane protein related projects.
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