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Multi-pass Transmembrane Proteins and Technology Platforms

Transmembrane Proteins Platform
INTRODUCTION
Multi-pass transmembrane target proteins
                    Figure 1.Multi-pass transmembrane target proteins
Transmembrane proteins (TPs) are embedded in the cell membrane and span both the intracellular and extracellular environments.The transmembrane region, which directly interacts with the phospholipid bilayer, is an important channel connecting these environment and enables transport of various ions and molecules as well as relaying activation and response reactions to extracellular stimuli. These reactions propagate intracellularly through downstream signal pathways and regulate cell metabolism, cellular activity and cellular fate. TPs can be classified structurally as either alpha-helical or beta-barrels and topologically as single-pass or multi-pass molecules with orientation of N and C terminus. Many human diseases are known to be associated with abnormal functions of these TPs and their role in variety of signaling pathways makes them ideal drug targets.
In order to develop effective therapeutic targeting these TPs, a full length and biologically relevant form of these proteins needs to be isolated. Particularly, multi-pass TPs are notoriously difficult to purify due to complex structures with multiple hydrophobic transmembrane regions and low expression level in host cells but have become highly sought-after disease target. It is confirmed that Rituximab recognizes CD20 not only at the primary epitope at the extracellular loop 2 (ECL2), but also its secondary epitope at extracellular loop 1 (ECL1).Furthermore, this secondary epitope also affects the CDC activity of Rituximab[1]. Therefore, a full length TPs with native folding is needed to fully characterize antibody activity and mechanism of action.
ACROBiosystems has specially set up multiple technology platforms to address the structural complexity of multi-pass TPs and meet the needs of different application. ACRO has developed a full range of full-length multi-pass TPs with stabilized structure and high activity including CD20, Claudin18.2, CD133, GPRC5D, CCR5 and CCR8(Figure1)to facilitate drug development and mechanism studies.
TECHNOLOGY PLATFORMS
VLP Platform
 VLP Platform
Principle

The VLP (Virus like particles) technology platform based on the HEK293 expression system is specially set up by ACROBiosystems to express TPs on the host cell surface. The viral envelop/capsid protein then turns these cell surfaces into soluble lipid bilayer particles with highly-concentrated proteins amenable for antibody immunization and screening. The membrane protein-VLP complex displays correctly folded multi-pass TPs in its cell membrane  induce and screen functional antibodies that recognize the natural conformation of the target.In addition to providing VLP based multi-pass TPs in our catalog, ACRO provides customized services as well.

Advantages
Full-length TPs with complete ECD epitopes
Higher abundance than that of overexpressing cells
Higher immunogenicity
Can be used as the best targets for dendritic cells and phage display in vivo because of their 100-200nm in size
Compatible with immunization/ELISA/SPR/BLI/cell experiment/CAR detection
Detergent Micelle Platform
Detergent Micelle Platform
Principle

The transmembrane region of multi-pass TPs is highly hydrophobic, and it is difficult to maintain the correct conformation in ordinary buffers once extracted from cell membrane. This problem can be solved by addition of detergents. ACROBiosystems has set up a complete platform for insect cell and mammalian cell expression, purification, and stabilization for difficult drug target TPs. ACRO performs detergent screening including DDM/CHS (Cat. No. DC-11) to increase solubility and ensure native fold of this protein in vitro.

Advantages
Full-length TPs, not only contain complete ECD epitopes
Can be accurately quantified
Compatible with immunization/ELISA/SPR/BLI
Nanodisc Platform
Nanodisc Platform
Principle

"Nanodisc" is a synthetic phospholipid bilayer membrane structure composed of membrane scaffold proteins (MSPs) and phospholipid molecules. TP can be integrated into the special structure of Nanodisc after removal of the detergent so as to maintains its native folding, retain its biological activity and exhibitimproved hydrophilicity for a wide range of applications. For example, the detergent free formulation of Nanodisc based TP is compatible in CAR expression tests. ACROBiosystems has licensed the Nanodisc technology from University of Illinois and performed continuous optimization and improvement of the assembly process to make it suitable for industrial scale-up production. ACRO’s efforts have ensured a long-term stable supply of Nanodisc based TP products for the biopharma industry.

Advantages
Full-length TPs are in a natural membrane environment retaining high biological activity
High hydrophilicity without detergents
Authorized licensed patent holder
Compatible with immunization/ELISA/SPR/BLI/cell experiment/CAR detection
PRODUCT LIST
  • VLP

  • Detergent Micelle

  • Nanodisc

MoleculeCat.No.Product DescriptionApplicationPreorder/Order
Claudin18.2CL2-H5547Human Claudin18.2 Full Length Protein-VLP (HEK293)Immunization
ELISA
SPR
BLI
Cell Experiment
CAR Detection

Order

GPRC5DGPD-H52P5Human GPRC5D Full Length Protein-VLP (HEK293)

Preorder

CCR5CC5-H52P3Human CCR5 Full Length Protein-VLP (HEK293)

Preorder

CCR8CC8-H52P4Human CCR8 Full Length Protein-VLP (HEK293)

Preorder

CD20 CDP-H52P6Human CD20 Full Length Protein-VLP (HEK293)

Order

VLPVLP-H52C5Virus-Like Particle (VLP) isotype controlIsotype Control

Order

MoleculeCat.No.Product DescriptionApplicationPreorder/Order
CD20CD0-H52H3Human CD20 / MS4A1 Full Length Protein, His Tag (HEK293) (SPR verified)Immunization
           ELISA
           SPR
           BLI

Order

CD20CD0-H82E5Biotinylated Human CD20 / MS4A1 Full Length Protein, His,Avitag™ (HEK293)

Order

CD20CD0-C52H8Cynomolgus CD20 / MS4A1 Full Length Protein, His Tag

Order

Claudin18.2CL2-H5546Human Claudin-18.2 Protein, His Tag (active membrane protein)

Order

Claudin18.2CL2-H82E3Biotinylated Human Claudin-18.2 Protein, His,Avitag™(active membrane protein)

Order

CD133CD3-H52H4Human CD133 Protein, His Tag

Order

CCR5CC5-H52D1Human CCR5 Protein, Flag,His Tag

Order

GPRC5DGPD-H52D3Human GPRC5D Protein, Flag,His Tag

Order

BufferDC-11200x DDM CHS bufferMaintain solubilization and activity of membrane proteins
           Dilute membrane proteins or antibodies
           Prepare running buffer in SPR or BLI assay, etc.

Order

MoleculeCat.No.Product DescriptionApplicationPreorder/Order
CD20CD0-H52H1Human CD20 / MS4A1 Full Length Protein, His Tag (Nanodisc) (HEK293)Immunization
           ELISA
           SPR
           BLI
           Cell Experiment
           CAR Detection

Order

CD20CD0-H82E3Biotinylated Human CD20 / MS4A1 Full Length Protein, His,Avitag™ (Nanodisc) (HEK293)

Order

CD133CD3-H52H1Human CD133 Full Length Protein, His Tag (Nanodisc)

Order

CD133CD3-H82E6Biotinylated Human CD133 Protein, His Tag (Nanodisc)

Preorder

GPRC5D GPD-H52D4Human GPRC5D Protein, Flag,His Tag (Nanodisc)

Order

MSP1D1 APO-H51H3Human MSP1D1 Protein, His Tag (Nanodisc)Isotype Control

Order

MSP1D1APO-H81Q5Biotinylated Human MSP1D1 Protein, His,Avitag™ (Nanodisc)Isotype Control

Order

Case Display

The following SPR protocols are available for free.

Claudin18.2-VLP

Correct assembly validated by SEM
Full-length Claudin18.2-VLP (Cat. No.CL2-H5547) has been observed under an electron microscope to ensure that it is assembled correctly.
Claudin18.2-VLP
Good bioactivity validated by ELISA
ELISA

Immobilized Human Claudin-18.2 Full Length Protein-VLP (Cat. No. CL2-H5547) at 5 μg/mL (100 μL/well) can bind Monoclonal Anti-Chimeric Claudin-18.2 Antibody, Human IgG1 with a linear range of 0.8-3 ng/mL (QC tested).       

High affinity validated by SPR
SPR

Human Claudin-18.2 Full Length Protein-VLP (Cat. No. CL2-H5547) captured on CM5 Chip via Anti-Claudin-18.2 antibody can bind Anti-Claudin-18.2 antibody with an affinity constant of 1.24 nM as determined in a SPR assay (Biacore T200) (Routinely tested).

CD20-DDM/CHS

Good bioactivity validation of full-length CD20-DDM/CHS(Cat. No.CD0-H52H3) by ELISA
Good bioactivity validation of full-length CD20-DDM/CHS

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Human CD20 Full Length, His Tag, HEK293 (SPR verified) (Cat. No. CD0-H52H3) with a linear range of 0.4-3 ng/mL (in presence of DDM and CHS).

Good bioactivity validation of CD20-DDM/CHS(Cat. No.CD0-H82E5) by ELISA
Good bioactivity validation of CD20-DDM/CHS

Immobilized Rituximab at 2 μg/mL (100 μL/well) can bind Biotinylated Human CD20 Full Length, His,Avitag (HEK293) (Cat. No. CD0-H82E5) with a linear range of 4-63 ng/mL (in presence of DDM and CHS).
                 

High affinity validation of CD20-DDM/CHS(Cat. No.CD0-H82E5) by SPR
High affinity validation of CD20-DDM/CHSE

Biotinylated Human CD20, His,Avitag (HEK293) (Cat. No. CD0-H82E5) captured on Biotin CAP-Series S Sensor Chip can bind Rituximab with an affinity constant of 1.73 nM as determined in a SPR assay (in presence of DDM and CHS) (Biacore T200).

CD133-Nanodisc

Purity greater than 90% by SDS-PAGE
Purity greater than 90% by SDS-PAGE

Human CD133 Full Length, His Tag (Nanodisc) ((Cat. No. CD3-H52H1))on SDS-PAGE under reducing (R) condition. The gel was stained overnight with Coomassie Blue. The purity of the protein is greater than 90%.
                  

Good bioactivity validated by ELISA
ELISA

Immobilized Human CD133 Full Length, His Tag (Nanodisc) (Cat. No. CD3-H52H1) at 1 μg/mL (100 μL/well) can bind Monoclonal Anti-Human CD133 (133-3) Antibody, Human IgG1 with a linear range of 0.2-4 ng/mL (QC tested).

Questions & Answers
Transmembrane Proteins Platform
Q1:For immunization and antibody screening, which platform should I choose for Claudin18.2?

Our Claudin18.2 has been developed with VLP and DMM/CHS platforms. Both of these versions are suitable for immunization and screening. However, the VLP version may be preferred for improved immunogenicity. For accurate affinity measurements, we recommend using the DDM/CHS version (the detergent version).

Q2:Why do you want to emphasize the importance of full-length protein? After all, antibodies and antigens only bind on the extracellular domain.

Multi-pass transmembrane proteins span the cell membrane multiple times forming multiple extracellular domains. For example, Claudin18.2, CD20, and CD133 have two extracellular loops (ECLs) with each ECL having specific functions and interactions with each other. Full length can ensure that the protein conformation is biologically relevant while enabling the ECL to be completely exposed for improved screening of ideal antibodies. As the figure below illustrates, the full length is active and relevant compared to isolated extracellular domain. It is not the case that ACRO always emphasizes full length proteins, but drug discovery R&D work needs the full-length multi-pass transmembrane proteins. Indeed, when compared, the activity of only the ECL region is worse than that of the full-length protein. ACRO is committed to providing the high quality and relevant products that meet customers' needs.

Structure of Full-length CD20 Protein
Q3:How do you exclude non-specific antibodies for the full-length membrane proteins in VLP and Nanodisc platforms?

The influence of non-specific antibodies cannot be ignored. Using membrane proteins under the VLP and Nanodisc platforms may produce non-specific antibodies. Both of our platforms have corresponding isotype controls for reverse-screening and excluding non-specific antibodies. Membrane protein-VLP has an isotype control product (Cat. No. VLP-H52C5). Membrane protein-Nanodisc has two isotype control products, one of which is MSP1D1(Cat. No. APO-H51H3) as the isotype control for tag free version. If a biotinylated membrane protein-Nanodisc is used, MSP1D1(Cat. No. APO-H81Q5) can be used as the isotype control.

VLP/Nanodisc isotype control products
Q4:Compared to other platforms, what are the advantages of Nanodiscs?

Nanodisc-membrane proteins are quite different from detergent stabilized membrane proteins. First of all, in principle, Nanodisc-membrane protein are assembled on native membrane-like structures and are completely detergent free. This opens up applications like immunization use for Nanodisc assembled proteins otherwise restricted due to detergent dissolving native cell membrane and causing damage to cells. In addition, the Nanodisc version of membrane proteins are compatible with cell-based assays and CAR expression detections. The Nanodisc platform used by ACRO has been authorized by the patent holder and can be used with confidence during the development process.

Q5:How do you confirm that Claudin18.2-VLP contains the target protein and how do you evaluate its purity? In the EM image on your product page, how do you tell whether the protein is embedded on the VLP particle?

Claudin18.2-VLP is tested and verified by anti-Claudin18.2 specific antibodies. The purity is evaluated by SDS-PAGE/HPLC/DLS/SEM. The conventional negative dye EM cannot see whether Claudin18.2 is on the VLP due to its low resolution. Binding activity with anti-Claudin18.2 specific antibodies can confirm the presence of Claudin18.2 on the particles.

Q6:Would VLP-membrane proteins be suitable for lyophilization? Could it be emulsified, and are there any requirements for the type of adjuvant? What are the precautions for using VLP formulated proteins for immunization?

We currently store all VLP formulated products as liquids at -70°C and ship on dry ice. We do store non- enveloped VLP products by freeze-drying. VLP immunization mainly requires attention to the choice of adjuvant dose, because VLP itself can enhance immunogenicity, which is not the case for conventional protein products.

Q7:You also have VLP versions of CD24 and PD-1. Are they the same as Claudin18.2? What is the difference between enveloped VLP and non-enveloped VLP?

CD24 and PD-1 are soluble proteins. They are not multi-pass transmembrane proteins, so non-enveloped VLPs are used. While multi-transmembrane proteins have hydrophobic transmembrane regions and need to be embedded on the membrane of the enveloped VLPs. The difference between VLP and enveloped VLP is whether there is a phospholipid bilayer membrane on the surface of the VLP.

Q8:How long is the lead time for customized full-length membrane protein?

If customized, the membrane protein in VLP format is expected to take 6-8 weeks.  The DDM/CHS version requires expression and purification conditions optimization of the target multi-pass transmembrane protein and detergent screening, so the development cycle is longer lasting more than 8 weeks. The customized Nanodisc version of membrane protein requires an additional 4 weeks on top of the DDM/CHS development time.

REFERENCES

Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab.

The role of CD133 in cancer: a concise review.

  • Authors: Paige M. Glumac , Aaron M. LeBeau.

  • Journal: Glumac and LeBeau Clin Trans Med

  • Download Full Article

Structural and Molecular Interactions of CCR5 Inhibitors with CCR5.

  • Authors: Kenji Maeda, Debananda Das et al.

  • Journal: Journal of Biological Chemistry

  • Download Full Article

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding.

  • Authors: Line Barington, Pia C. Rummel, et al.

  • Journal: Journal of Biological Chemistry

  • Download Full Article

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