A Simple Method for Improving Protein Solubility and Long-Term Stability

Abstract

Increasing a protein concentration in solution to the required level, without causing aggregation and precipitation is often a challenging but important task, especially in the field of structural biology; as little as 20% of nonmembrane proteins have been found to be suitable candidates for structural studies predominantly due to poor protein solubility. We demonstrate here that simultaneous addition of charged amino acids L-Arg and L-Glu at 50 mM to the buffer can dramatically increase the maximum achievable concentration of soluble protein (up to 8.7 times). These amino acids are effective in preventing protein aggregation and precipitation, and they dramatically increase the long-term stability of the sample; additionally, they protect protein samples from proteolytic degradation. Specific protein-protein and protein-RNA interactions are not adversely affected by the presence of these amino acids. These additives are particularly suitable for situations where high protein concentration and long-term stability are required, including solution-state studies of isotopically labeled proteins by NMR.

Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins

“…It has been speculated that it has a completely different origin from other RE 359 effects, and is related to competitive inhibition of contaminating proteases by the 360 added amino acids (Golovanov et al 2004). 361…”

“…The solubility of glutamic acid can be increased to much higher 457 concentrations in the presence of equimolar amounts of arginine. The increase in 458 protein solubility in 50 mM RE equimolar mixture is not observed in the presence 459 of the same concentration of the individual amino acids (Golovanov et al 2004). 460 RE also has a larger effect than arginine on the second virial coefficient of 461 lysozyme (Valente et al 2005).…”

“…by Golovanov et al (2004) to prevent aggregation and to improve the solubility of 54 a large number of proteins while preserving biologically relevant macromolecular 55 interactions. The same publication reported an increased resistance to 56 degradation by proteases in the presence of 50 mM RE.…”

Fast infrared spectroscopy of protein dynamics: advancing sensitivity and selectivity

“…As a guideline of what is required, we note that NMR spectroscopy typically works at concentrations around 0.5 mM, but even in that regime only about 20% of nonmembrane proteins remain soluble [15]. The likelihood of a given protein to aggregate increases very steeply with concentration.…”

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…Arginine/glutamate buffer is a promising buffer not only because it can increase protein stability and solubility [17,162] but it also results in better signal to noise ratios in cryogenic probes due to its lower ionic strength compared to other buffers [163]. However, the disadvantage is that this buffer causes baseline distortions due to its strong NMR signals.…”