共振光散射法可直接表征蛋白质的溶解度  

Resonance light scattering spectroscopy can directly characterize protein solubility

作　　者：陈童 同婷婷 杨林玉 廖飞 杨晓兰[1] 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)

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016 [2]重庆理工大学药学与生物工程学院,重庆401135

出　　处：《南方医科大学学报》2020年第6期843-849,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(81773625,31570862,30672009);重庆市自然科学基金项目(CSTC2019jcyj-msxmX0166)。

摘　　要：目的建立一种快捷、灵敏、成本低的蛋白质溶解度的共振光散射(RLS)表征方法,用于蛋白质突变体溶解度比较。方法同步扫描模式下,跟踪RLS信号强度对蛋白浓度的响应,根据响应曲线拐点预测蛋白质相应分散状态的最大浓度,据此定义该状态的溶解度。选择牛血清白蛋白(BSA,0~50.0 g/L),考察pH(6.5、7.0、7.4)、盐离子浓度(0.05、0.10、0.15、0.20 mol/L)对溶解度测定值的影响;并测定谷胱甘肽S-转移酶同工酶alpha(GSTA,0~27.0 g/L)、Mμ(GSTM,0~20.0 g/L),考察方法通用性。应用RLS测定季也蒙毕赤酵母菌尿酸酶(MGU,0~0.4 g/L)溶解度并指导其突变体溶解度改进。结果所测蛋白质RLS响应曲线均出现两个拐点,低浓度拐点可近似蛋白质单分子分散状态的最大浓度,而高浓度拐点近似蛋白质多分子聚集状态的最大饱和浓度即热力学平衡溶解度。BSA(等电点4.6)两个拐点均随pH的增大(6.5~7.4)而增高,pH7.4 HEPES缓冲液中分别对应~1.2 g/L、~33 g/L,后者与相同条件下商品溶解度接近;加入NaCl使两个浓度拐点均降低,初步表明RLS可直接表征蛋白溶解度。GSTA、GSTM的RLS响应曲线低浓度拐点为~0.7 g/L,~0.8 g/L,高浓度拐点分别为~10 g/L、~11 g/L,均小于BSA,方法有通用性。MGU两个拐点分别对应0.24 g/L和0.30 g/L,而其突变体溶解度提高约2倍。结论共振光散射法可直接表征蛋白质溶解度,且简便、灵敏,蛋白用量少,尤其适用于表达丰度低的蛋白质突变体溶解度的快速比较。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

关 键 词：蛋白质 溶解度 共振光散射 尿酸酶 

分 类 号：R446.1[医药卫生—诊断学]