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Protein Processing - NVoy Technology

Protein Processing - NVoy Technology

Protein processing and production is often hampered by the formation of aggregates that restrict and complicate the handling of proteins, antibodies and enzymes. NVoy polymers are designed to minimize the sequential losses in consecutive protein processing steps which would otherwise dramatically reduce the overall protein yield.

NVoy technology is an alternative to the use of detergents, fusion proteins, arginine, chaperones and a range of other common additives employed to increase protein solubility and enable the handling of proteins in solution for better performance in downstream applications (MS, crystallization, HTS assays, etc.).

NVoy technology utilizes NV polymers (NV10), proprietary carbohydrate polymers, which are designed to increase the solubility and stability of proteins while preventing aggregation and reducing non-specific binding. NV polymers are linear, uncharged carbohydrate polymers of around 5kD, derivatized to make them highly amphipathic. They associate at multiple points with surface exposed hydrophobic patches of proteins in a dynamic fashion to form multipoint, reversible complexes. Multiple-binding points allow NV polymers to be used at low concentrations relative to alternative reagents, and their size prevents them from entering the protein core and inhibiting normal structural bonding or blocking catalytic/binding sites. NV polymers are easily separated from the protein when they are no longer required in solution.

Applications

Protein Stabilization

While stability is very protein specific, NV polymers will typically protect and stabilize target proteins. 

  • Maintain activity over several freeze/thaw cycles
  • Prolonged storage at 4ºC for unstable proteins
  • Maintain solubility of fusion proteins after “tag” is removed
  • Stable formulation of protein / antibody for immobilization and conjugation
  • Maintain soluble proteins that usually require the presence of a ligand
  • Increase concentration of proteins that would otherwise aggregate when concentrated

nvstability

Figure 1 (left): Within 5 hours of preparing a set of citrate synthase standards, the standard curve has lost linearity and the activities of the standards at 0.4 μg/ml and 1.1 μg/ml have virtually disappeared. Figure 2 (right): The citrate synthase standard curve produced from samples containing NV10 remains linear even after 96 hours.

Protein Concentration

NVoy technology can be used to minimize protein losses due to aggregation and non-specific binding during concentration. NV10 will co-concentrate with the protein in solution to give continuing protection.

nvconc

Table: Duplicate samples containing 10 μg/ml BSA in PBS and supplemented with varying concentrations of NV10 were concentrated tenfold in Vivaspin 2 spin concentrators (5,000 MWCO, Hydrosart low protein-binding membrane) according to the manufacturer’s instructions.

Aggregate Reduction

Protein processing and production is often hampered by the formation of aggregates. NVoy technology is an alternative to the use of detergents, fusion proteins, arginine, chaperones and a range of other common additives employed to increase protein solubility and handling. The NVoy polymer increases the solubility and stability of proteins while preventing aggregation and reducing non-specific binding.

Fusion Protein ‘tag-removal’

NV10 associates with the protein in solution and protects the cleaved native protein from aggregation and instability without affecting the cleavage reaction.

nvfusion

Figure: Cleavage of the protein kinase fusion CK2alpha/-MBP. Removal of the maltose binding protein fusion partner (k-MBP) from the kinase, using Factor Xa, results in heavy aggregation and low yields of the native kinase (far left - no NV10). By adding NV10 to the cleavage buffer, aggregation was significantly reduced while the cleavage reaction remained unaffected.

Protein Refolding

Addition of NV10 to refolding buffer and throughout the refolding protocol helps suppress protein aggregation and may allow refolding at final protein concentrations greater than 0.25 mg/ml.

Endotoxin Removal

Use of NVoy polymers during affinity chromatography or ion exchange of the target protein assists in the release of endotoxin prior to protein elution.

nvendotoxin

Table: Comparison of Endotoxin removal methods. Target protein was bound to an affinity resin and processed according the NVoy procedure, the Detoxi Gel procedure (Pierce) and the method by Reichelt et al. using Triton X114 (Reichelt et al., Protein Expression and Purification, 46 (2006) pp.483-488). NVoy assisted endotoxin removal was the only method that resulted in a final endotoxin concentration below the detection limit. Moreover, the use of NVoy resulted in significantly higher protein recovery and protein activity.

Protein Processing & Purification

Use of NV10 results in cleaner, active protein preparations compared to the use of detergents and enhances protein recovery, stability and storage.

nvclean

Figure: Comparision of protein processing using NV10 vs. detergents.

Crystallization

Because NVoy polymers improve protein solubility and prevents aggregation, it offers benefits for many downstream applications like protein crystallization.

  • Controllable crystal growth when rapid formation produces poor crystals
  • Concentrate and maintain high protein concentration without the need for any other additives
  • Solubilize and stabilize proteins for longer periods of time

 

Ordering

Product

NVoy Stabil-PAC Kit

NVoy Polymer Pack

Size1

6 x 10 mg

1.25 grams

Size2

6 x 40 mg

5 grams