Detection and prevention of protein aggregation before, during, and after purification

Abstract

The use of proteins for in vitro studies or as therapeutic agents is frequently hampered by protein aggregation during expression, purification, storage, or transfer into requisite assay buffers. A large number of potential protein stabilizers are available, but determining which are appropriate can take days or weeks. We developed a solubility assay to determine the best cosolvent for a given protein that requires very little protein and only a few hours to complete. This technique separates native protein from soluble and insoluble aggregates by filtration and detects both forms of protein by SDS-PAGE or Western blotting. Multiple buffers can be simultaneously screened to determine conditions that enhance protein solubility. The behavior of a single protein in mixtures and crude lysates can be analyzed with this technique, allowing testing prior to and throughout protein purification. Aggregated proteins can also be assayed for conditions that will stabilize native protein, which can then be used to improve subsequent purifications. This solubility assay was tested using both prokaryotic and eukaryotic proteins that range in size from 17 to 150 kDa and include monomeric and multimeric proteins. From the results presented, this technique can be applied to a variety of proteins.

In Pursuit of Synthetic Modulators for the Orphan Retina-Specific Nuclear Receptor NR2E3

“…To overcome aggregation that leads to purification of the inactive protein, we tested a variety of agents that are known to either destabilize aggregates or increase native protein stability. 29 Protein aggregation was assayed based on separation of aggregated and soluble NR2E3 using the Nanosep 300K Omega centrifugal device, as described in the Methods section. As shown in Fig.…”

Hox Transcription Factor Ultrabithorax Ib Physically and Genetically Interacts with Disconnected Interacting Protein 1, a Double-stranded RNA-binding Protein

“…Biotinylated Ubx Ib was expressed in E. coli AR120. Cell growth and protein induction were performed as described (32). Cell pellets from a 200-ml growth were resuspended in 2 ml of 10% sucrose, 50 mM Tris-HCl, pH 7.4, with 0.5 mM PMSF and 0.8 mg/ml lysozyme and frozen at Ϫ80°C.…”

“…All buffers contained 5% glucose to minimize precipitation of DIP1-c as established by an aggregation assay (32). DIP1-c runs at an anomolously high molecular weight on SDS-PAGE, a trait previously reported for other proteins containing dsRNA-binding domains (38,43).…”

A hybrid neutron diffraction and computer simulation study on the solvation of N-methylformamide in dimethylsulfoxide

“…They are important in the control of protein denaturation 1 and its absence may be responsible for spurious aggregation of proteins. 2,3 Understanding the interactions between small molecules and how they associate with each other may be useful for understanding the binding of small biomolecules in solution and its influence on the self-assembly of larger functionally active biological molecules. The influence of dimethylsulfoxide (DMSO) on the peptide behavior is an interesting case of chaotropy/kosmotropy.…”

“…The SO polar group a) Author to whom correspondence should be addressed. can give rise to H-bond formation, while the two CH 3 groups can bring about hydrophobic effects. 23 N-methylformamide (NMF), on the other hand, contains the peptide bond in its structure and can act as proton donor and acceptor via its C=O and N-H groups and consequently form C=O· · · H-N hydrogen bonds (H-bond) to each other, the same type of H-bond that is known to play an important role in the stabilization of the ordered intramolecular structure of peptides and proteins in aqueous medium.…”