NVoy minimises the sequential loss of protein in consecutive processing steps
Utilising 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. The NV10 polymer can be bought as part of kits such as Stabil-P.A.C and the refold kits or loose in 1.25 gram or large pack sizes. The polymer is designed to increase the solubility and stability of proteins whilst preventing aggregation and reducing non-specific binding.
NVoy polymers are linear, uncharged carbohydrate polymers of around 5kD, derivatised 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. Based on simple carbohydrate polymers, NV polymers are easily separated from the protein when they are no longer required in solution.
Impact Areas of NVoy Technology
The impact of NVoy technology can be seen in many areas of protein research including stabilisation, purification, analysis and crystallisation.
Maintain activity over several freeze/thaw cycles, Prolonged storage at 4C for unstable proteins
Maintain solubility of fusion proteins after “tag” is removed
Stable formulation of protein/antibody for immobilisation + conjugation
Maintain soluble proteins that usually require ligand to be present
Increase concentration of proteins that would otherwise aggregate when concentrated
Improve purification strategy
Minimise protein losses
Cleaner protein preparations
Allow full structural characterisation (MS, crystallisation, NMR, CD)
Use in HTS assays to keep proteins soluble and reduce non-specific binding
Replacing detergents which are more difficult to remove downstream
Enhance solubility enabling concentration of aggregation prone proteins
Prevent non-specific binding to plastic and hydrophobic membranes
Concentrate and maintain high protein concentration without the need for any other additives
Controllable crystal growth when rapid formation produces poor crystals
Solubilise and stabilise proteins for longer period of time
Simple, effective, generic technique
Figure 1: 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: The citrate synthase standard curve produced from samples containing NV10 remains linear even after 96 hours.
Concentration of Bovine Serum Albumin
Duplicate samples containing 10 μg/ml BSA in PBS and supplementedwith varying concentrations of NV10 were concentrated tenfold in Vivaspin2 spin concentrators (5,000 mwco, Hydrosart low protein-binding membrane) according to the manufacturer’s instructions.
|Starting solution (1 ml)||Recovered yield (%)|
|10 μg/ml BSA||46%|
|10 μg/ml BSA + 10 μg/ml NV10||60%|
|10 μg/ml BSA + 40 μg/ml NV10||85%|
|10 μg/ml BSA + 100 μg/ml NV10||90%|
Target protein was bound to an affinity resin and washed with 10 column volumes (CV) of wash buffer containing 2% NV10 followed by 10 CV wash buffer containing 0.2% NV10 followed by 5 CV wash buffer. Target protein was eluted and analysed for endotoxin content. The same protein was processed with Detoxi Gel procedure (Pierce) according to the manufactures instruction.
NVoy assisted endotoxin removal was the resulted in a final endotoxin concentration below the detection limit. Moreover the Expedeon method resulted in significantly higher protein recovery and protein activity.
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. By adding NV10 to the cleavage buffer (20 mM Tris.HCl, 75 mM NaCl, 1 mM CaCl2, pH 6.5) aggregation can be significantly reduced, whilst the cleavage reaction remains unaffected. Cleavage of the protein kinase fusion CK2α/-MBP
Each Stabil-PAC kit contains NVoy technology designed to increase the solubility and stability of proteins whilst preventing aggregation and reducing non-specific binding. Stabil-PAC kits are a convenient way to test if NVoy is able to assist your work. Containing single shot amounts of lyophilised NVoy in six aliquots, ensuring reagent integrity. Once the initial assessment has been made using these kits, larger quantities can be obtained to satisfy your project requirements.
Expedeon offers a range of protein refolding kits that provide a simple, generic method for refolding target proteins without the need to screen multiple refolding conditions. NVoy Protein Refolding Kits often produce higher yields of fully refolded protein as NVoy protects the vulnerable intermediates in the refolding process. Furthermore, with NVoy being removable in a slow, time dependant and user controllable manner, the protein is provided with sufficient time and protection to refold correctly.
NVoy Polymer Packs
Large quantities of NVoy reagent enable cost effective scaling of NVoy stabilisation. Pack sizes range from 1.25g upwards. Contact us for further information.