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 can be tailored to your specific needs. Download our service brochure to learn more.

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 other fail! 95 % success rate for membrane protein expression and stabilization!	Internationally approved! Over 30 projects with „Big Pharma“ completed!

An example of our typical workflow can be seen here. Download it!

Fig. 1: Typical workflow to produce your protein of interest. Contact us to discuss your project with us!

Our service introduction video - learn why we are your best option for membrane protein related projects.

Download our service brochure as a PDF!

Our modules in detail (Click on the green arrow)

Module 1: Protein Expression

As a first step we determine the right expression system for your protein. We use the Baculovirus expression system in Sf9 or T. ni (High FiveTM) insect cells (up to 50 L scale). Furthermore we use the BacMam system 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.

Expression system	Advantages	Amount of purified protein
Cell free expression system:	For direct stabilization and co-translational integration of membrane proteins into nanodiscs For toxic proteins	Highest among our expression systems.
Baculovirus Insect system	Near mammalian like post-translational modifications Easy to handle eucaryotic expression system.	Higher than mammalian cell lines
Hek293 cells	Homologue mammalian protein expression.	Lowest of the three, but with highest authenticity among our 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:

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: We screen for the best detergent for your membrane protein. This procedure belongs to the daily buisness of our lab staff. Detergent screening parameters are:

Detergent Concentration
Solubilization time
Buffer composition

Synthetic Polymers:

New substances like SMALPS and DIBMA dissolve the cell membrane. They simultaneously solulbilize and stabilize a membrane protein. Therefore using these synthetic polymers combines module 2 and 3 into one step.

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.

Membrane protein surrounded by detergents

Figure 2: Membrane protein (orange) stabilized through detergents (black).

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.

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.
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.