Chen Tong, Tong Tingting, Yang Linyu, Liao Fei, Yang Xiaolan
Key Laboratory of Medical Laboratory Diagnostics of the Ministry of Education of China, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
School of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing 401135, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2020 Jun 30;40(6):843-849. doi: 10.12122/j.issn.1673-4254.2020.06.11.
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.
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.
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.
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.
开发一种基于共振光散射（RLS）的快速、灵敏且经济高效的方法，用于表征蛋白质溶解度，以促进对突变蛋白溶解度变化的检测。
我们在同步扫描模式下研究了RLS强度对蛋白质浓度的响应曲线。搜索曲线交点以预测处于分散状态的蛋白质的最大浓度，该浓度定义了蛋白质在给定状态下的溶解度。以牛血清白蛋白（BSA，0 - 50 g/L）为模型，研究pH值（6.5、7.0和7.4）和盐浓度（0.05、0.10、0.15和0.20 mol/L）对测定溶解度的影响。估算谷胱甘肽S - 转移酶同工酶α（GSTA，0 - 27.0 g/L）和Mμ（GSTM，0 - 20.0 g/L）的溶解度以作比较。使用基于RLS的方法测定尿酸酶（MGU，0 - 0.4 g/L）的溶解度，以辅助提高其突变体的溶解度。
我们在测试蛋白质的RLS响应曲线中识别出两个交点，其中较低的交点代表单分子分散状态下蛋白质的最大浓度（或溶解度）的近似值，较高的交点代表多分子聚集体中蛋白质的饱和浓度。在HEPES缓冲液中，BSA（等电点4.6）的两个交点均随pH值升高（6.5 - 7.4）而增加，在pH 7.4时其值分别约为1.2 g/L和约33 g/L；后者浓度接近相同缓冲液中相同pH下市售BSA的溶解度。添加NaCl降低了两个交点的值，且增加盐离子浓度会降低较低交点的值。对GSTA和GSTM的进一步表征表明，这两种蛋白质的低浓度交点分别约为0.7 g/L和约0.8 g/L，高浓度交点分别约为10 g/L和约11 g/L，均低于BSA，表明通过RLS直接表征蛋白质溶解度的可行性。MGU的两个浓度交点分别为0.24 g/L和0.30 g/L，其选定突变体的低浓度交点增加了2倍。
RLS可直接表征大分子蛋白质的溶解度。该方法简单、灵敏，仅需少量蛋白质，在快速比较低丰度蛋白质突变体的溶解度方面具有独特优势。
