The modular structure of our Protein Production Services

Module 1: Protein Expression

As a first step we determine the right expression system for your protein or multimeric protein complex. 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.

Cell free expression system

For direct stabilization and co-translational integration of membrane proteins into nanodiscs
For toxic proteins
Highest yield

Baculovirus Insect system

Near mammalian like post-translational modifications
Easy to handle eucaryotic expression system.
Higher yield than mammalian cell lines

Hek293 cells

Homologue mammalian protein
Highest Authenticity
Lowest Yield

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 Expression

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:

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 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 choiceof 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.

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!

Module 4: Protein Purification

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

Figure 3: A membrane protein stabilized inside a MSP nanodisc.

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).

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.

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.

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.

Figure 5: Cube Biotech's PureCube INDIGO-NI resin inside a column ready for protein purification.

Module 5: Protein Characterization

Figure 6: Custom Resins use a 100% specific affinity towards your protein of interest.

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

Module 6: Protein Structure Determination

Figure 6: Custom Resins use a 100% specific affinity towards your protein of interest.

One of the key characteristics of a protein is its 3D structure. Unfortunately it is also one of the hardest characteristics to identify. NC PROBIOTECH 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 which combines the lipid cubic phase and vapor diffusion. Therefore we have gathered lots of experience with this method.

Module 7: Production Scale-Up

Figure 8: Cryo-Em has one of the best resolutions for protein structures to date!

Figure 9: Protein crystal in cubic phase

Benchtop to industrial scale production is hard to develop. Fortunately our team of scientists can help. Our scientists have:

Experience developing and implementing bench-scale protein purification methods for processing of raw mammalian cell culture harvest in biopharmaceutical laboratory environment.
Applied experience in protein purification methods including Affinity Chromatography, SEC, HIC, IEX, filtration/ultrafiltration, resin selection, and dealing with HMW and LMW species.
Technology transfer and implementation of bench-scale process at pilot and large-scale for GMP manufacturing.