[1]陈 童,同婷婷,杨林玉,等.共振光散射法可直接表征蛋白质的溶解度[J].南方医科大学学报,2020,(06):843-849.[doi:10.12122/j.issn.1673-4254.2020.06.11]
 Resonance light scattering spectroscopy can directly characterize protein solubility[J].Journal of Southern Medical University,2020,(06):843-849.[doi:10.12122/j.issn.1673-4254.2020.06.11]
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共振光散射法可直接表征蛋白质的溶解度()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2020年06期
页码:
843-849
栏目:
出版日期:
2020-06-17

文章信息/Info

Title:
Resonance light scattering spectroscopy can directly characterize protein solubility
作者:
陈 童同婷婷杨林玉廖 飞杨晓兰
关键词:
蛋白质溶解度共振光散射尿酸酶
Keywords:
protein solubility resonance light scattering uricase
DOI:
10.12122/j.issn.1673-4254.2020.06.11
文献标志码:
A
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。
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. Conclusion 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.

相似文献/References:

[1]谭小丹,卢智勇,苏永春,等.蛋白质中变构通讯结构基础的方法分析与实现[J].南方医科大学学报,2005,(06):675.
 TAN Xiao-dan,LU Zhi-yong,SU Yong-chun,et al.Analysis and realization of the method for predicting physical pathways of allosteric communi-cation in a protein family[J].Journal of Southern Medical University,2005,(06):675.

更新日期/Last Update: 2020-06-17