Resonance light scattering spectroscopy can directly characterize protein solubility.
10.12122/j.issn.1673-4254.2020.06.11
- Author:
Tong CHEN
1
;
Tingting TONG
1
;
Linyu YANG
1
;
Fei LIAO
2
;
Xiaolan YANG
1
Author Information
1. Key Laboratory of Medical Laboratory Diagnostics of the Ministry of Education of China, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
2. School of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing 401135, China.
- Publication Type:Journal Article
- Keywords:
protein;
resonance light;
solubility;
uricase
- MeSH:
Hydrogen-Ion Concentration;
Light;
Scattering, Radiation;
Solubility;
Spectrum Analysis
- From:
Journal of Southern Medical University
2020;40(6):843-849
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To develop a fast, sensitive and cost-effective method based on resonance light scattering (RLS) for characterization of protein solubility to facilitate detection of changes in solubility of mutant proteins.
METHODS:We examined the response curve of RLS intensities to the protein concentrations in synchronous scanning mode. The curve intersection points were searched to predict the maximal concentrations of the protein in dispersion state, which defined the solubility of the protein in this given state. Bovine serum albumin (BSA, 0-50 g/L) was used as the model to investigate the influences of pH values (6.5, 7.0, and 7.4) and salt concentrations (0.05, 0.10, 0.15, and 0.20 mol/L) on the determined solubility. The solubility of glutathione S-transferase isoenzymes alpha (GSTA, 0-27.0 g/L) and Mμ (GSTM, 0-20.0 g/L) were estimated for comparison. The RLS-based method was used to determine the solubility of uricase (MGU, 0-0.4 g/L) to provide assistance in improving the solubility of its mutants.
RESULTS:We identified two intersection points in the RLS response curves of the tested proteins, among which the lower one represented an approximation of the maximal concentration (or the solubility of the protein) in single molecular dispersion, and the higher one the saturated concentration of the protein in multiple molecular aggregation. In HEPES buffer, the two intersection points of BSA (isoelectric point 4.6) both increased with the increase of pH (6.5-7.4), and their values were ~1.2 g/L and ~33 g/L at pH 7.4, respectively; the latter concentration approached the solubility of commercial BSA in the same buffer at the same pH. The addition of NaCl reduced the values of the two intersection points, and increasing salt ion concentration decreased the values of the lower intersection points. Further characterizations of GSTA and GSTM showed that the low concentration intersection points of the two proteins were ~0.7 g/L and ~0.8 g/L, and their high concentration intersection points were ~10 g/L and ~11 g/L, respectively, both lower than those of BSA, indicating the feasibility of the direct characterization of protein solubility by RLS. The two concentration intersection points of MGU were 0.24 g/L and 0.30 g/L, respectively, and the low concentration intersection point of its selected mutant was increased by 2 times.
CONCLUSIONS:RLS allows direct characterization of the solubility of macromolecular proteins. This method, which is simple and sensitive and needs only a small amount of proteins, has a unique advantage for rapid comparison of solubility of low-abundance protein mutants.
