CN103608351A - 纯白蛋白及其制备和检测方法 - Google Patents
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Abstract
本发明的主题在于纯的单体牛血清白蛋白、特征为在树脂中使用柱色谱法的制备该纯的单体牛血清白蛋白的方法以及使用动态光散射鉴定该纯的单体牛血清白蛋白的方法。
Description
牛血清白蛋白(BSA)是最便宜的可商购的蛋白之一。其在纯理论研究[1-4]和大量实际使用[5-18]中广泛用作模型蛋白。得自BSA水解的游离氨基酸的混合物通常用于测定蛋白的氨基酸含量。用于该目的的商品BSA是足够纯的，尽管存在被糖污染的可能性。由于水解，污染物被降解且不妨碍氨基酸分析。白蛋白是循环系统中脂肪酸和其它疏水性物质的转运体。由此，在将BSA用于研究之前，有必要使用炭除去疏水性污染物。BSA用于检测GFP(绿色荧光蛋白)，促进被评估的细胞的超灵敏成像[6]。与量子点结合的修饰的BSA表面已经用于金属离子的分析检测[7]。因为该蛋白已成为光动力学疗法中光敏剂的有效载体，所以其可以用作炎症和肿瘤的治疗中有效成分的转运体[9-12]。此外，作为小分子的载体，BSA增加了所附着的化合物的免疫原性，这可能获得用于在ELISA测试中检测小分子的高灵敏性和特异性抗体[13,14]。BSA还用于构建结合疫苗。在与多糖抗原结合之后，可以获得抗病原体的有效且安全的疫苗。在类似的系统中，BSA还用于肿瘤治疗[15-18]。在这样的用途中，BSA应具有高纯度。特别地，其应没有糖污染。早期的报道指出在BSA制剂中存在高分子量衍生物[19]。可以形成高度聚合的聚集体，这是因为蛋白与反应性羰基化合物通过糖化而反应[20-23]。由于在蛋白环境中存在还原性糖，因此形成这样的污染聚集体可能存在风险。一系列报道表明在干燥条件下在热修饰期间以及在干粉制剂中，蛋白被果糖、葡萄糖、半乳糖、乳糖和葡聚糖糖化[24-27]。Fischer等人分析了各种治疗性单克隆抗体在其储存期间在含有具有糖组分的缓冲剂的制剂中以及在这些蛋白与葡聚糖在输液袋(通常用于给予治疗性蛋白的临床实践中)中进行短期孵育之后的糖化产物[28]。已经观察到商品BSA的自然糖化[19]。白蛋白易于在体内被糖化为大部分常见的血清蛋白[29,30]。在干燥条件下在高温合成期间，在BSA与己醛糖的反应期间也已经观察到聚集[31]。在BSA与葡萄糖和果糖一起孵育之后，已检测到高度糖化产物[25,29,31,32]。糖污染的商品BSA制剂的GLC-MS分析已经显示aldithiol acetate(多羟糖醇乙酸酯)的存在，这表明存在着摩尔比分别为1:1.6:1的甘露糖、葡萄糖和半乳糖。在早期报道中描述了在BSA与乳糖一起孵育之后在BSA中出现半乳糖[26]。已知各种方法能分离蛋白，利用蛋白分子大小[19]、表面电荷[33]以及对给定载体的亲和性[34]。不存在已知的从商品BSA制剂中分离纯的单体白蛋白的方法。尽管在由琼脂糖凝胶电泳测定的试剂特性中显示出98%的纯度，但SDS/PAGE变性电泳证实[35]具有更高分子量的蛋白衍生物。因此，优选通过SDS/PAGE测定BSA制剂的纯度。在商购制剂Cohn fractionV(Sigma,A7888,Poland)中已知高分子量形式[19]。已经描述BSA单体与这样的高分子量蛋白聚集体的分离，其利用在室温下，在装有葡聚糖凝胶Sephadex G-200(Pharmacia,Sweden)的、用PBS(磷酸盐缓冲盐水)(pH7.5，含0.02%NaN3)平衡的柱中进行分级[19]。然而，证实了0.25%的糖污染[19]。因此，使用Sephadex G-200作为分子筛不是优选的，因为多糖对分离的蛋白的污染水平增加，这归因于来自凝胶、蛋白修饰和其它反应的葡聚糖分子的参与。根据早期报道，BSA可以由葡聚糖通过Maillard反应进行修饰，并形成各种分子量的BSA-葡聚糖轭合物[36]。在可商购的BSA制剂中糖轭合物和高分子质量衍生物的存在使该制剂从分子生物学的主要研究应用中被排除。为此，必须设计从商购制剂中获得单体形式且没有糖内含物的BSA的方法。出人意料地，本发明解决了前述问题。
本发明的第一主题为分析纯的单体牛血清白蛋白，其不含在12%分离凝胶中通过SDS/PAGE用电泳测定的糖污染物。我们由分析纯来表示通过前述方法检测的不存在糖污染物。同样优选地，本发明的纯的单体牛血清白蛋白特征在于分子直径基本为7nm。
本发明的第二主题为分离单体血清白蛋白的方法，其特征在于所述方法包括：
a)制备BSA样品，
b)在树脂上色谱纯化从阶段(a)获得的样品，所述树脂优选为用平衡缓冲剂平衡的HW-55、所述平衡缓冲剂优选为0.1M的pH5.65的含1%正丁醇的乙酸盐缓冲液。同样优选地，本发明的方法特征在于阶段a)包括去除疏水性配体，优选通过使样品经过炭来去除。同样优选地，本发明的方法特征在于阶段b)中的纯化在室温下以1.2ml/10min的速率发生。
本发明的第三主题为鉴定白蛋白的方法，其特征在于所述方法包括在使用本发明的方法获得的级份(fractions)中使用动态光散射来测定蛋白分子的直径，其中单体级份为显露出直径基本为7nm的分子的级份。
根据本发明获得的BSA的单体级份的主要优势为所得蛋白的同质性(homogeneity)和不存在糖污染物。通过动态光散射(DLS)测定从HW-55S柱中获得的级份中的蛋白分子直径，从而另外确认了使用常规的变性电泳法测定的所得蛋白的同质性[37]。分子直径的值确认了单体BSA的存在且符合先前文献的数据[38]。通过DLS鉴定所得BSA单体是优选的，这归因于同时测定所得白蛋白级份的同质性(%)的可能性。明确的优势在于与通过SDS/PAGE的常规纯度分析相比时间降低。优选使用酸酚法[39]来测定所获得的BSA单体级份的总糖含量。在所获得的制剂中不存在糖污染物证明了为本发明主题的方法与两种其它分离单体牛白蛋白(在实例中描述为不利的)的方法相比具有优势。根据本发明从商购制剂分离BSA单体的另外优势为程序简单且成本低。
本发明的例示实施方案在图示中示出，其中图1表示商购BSA(Sigma-Aldrich A3294)的洗脱图谱：A)采用HW-55S上的色谱法；B)采用Sephadex G-200上的凝胶过滤；C)DEAE-Sephadex A-50上的阴离子交换色谱法；图2表示在12%分离凝胶中BSA样品的SDS/PAGE分析，其中泳道(lane)1)蛋白分子量标记物标准；2)6μg商购BSA样品(Sigma-Aldrich A3294)；泳道3)和4)分别为采用HW-55S色谱法获得的3μg级份A和级份B；泳道5)从HW-55S柱洗脱的6μg级份；泳道6)和7)分别为采用Sephadex G-200柱的2μg级份A和级份B；泳道8)采用Sephadex G-200中分级而获得的3μg级份C；泳道9)、10)和11)分别为采用在DEAE-Sephadex A-50上进行纯化的2μg级份90-100、115-123和124-150；图3表示蛋白分子直径的动态光散射(DLS)分析和通过HW-55S级份的纯化色谱法而获得的牛白蛋白的纯度水平。A)从柱中洗脱的蛋白分子的直径：具有黑色三角形的实线表示吸收率A280(作为级份中蛋白含量的量度)，具有黑色正方形的实线表示分子的直径，间断线表示7nm的直径(作为牛白蛋白的参照)。B)测定蛋白级份的纯度水平(以%计)：级份58-60的灰色柱表示具有参照牛白蛋白直径(7nm)和100%纯度的蛋白，图4表示具有核心寡糖的BSA修饰产物(用200倍摩尔过量的寡糖在37℃历时16小时而进行的蛋白修饰)的在12%SDS/PAGE凝胶中的电泳：泳道1-蛋白重量标准；2-用寡糖孵育的商购BSA；泳道3和4-分别为在没有寡糖的情况下在相同条件下孵育的商购和单体BSA的对照样品(采用在HW55-S柱上进行纯化而获得的)；泳道5-用寡糖修饰的BSA单体。
根据从商购Sigma-Aldrich制剂中(Conn fraction V,A3294,超过98%的纯度且使用琼脂糖凝胶电泳由生产商测定)分离BSA单体的实例，在例示实施方案中显示出本发明的主题。在分级色谱法之前，使商购的BSA样品通过炭以除去潜在的疏水性配体，主要为脂肪酸。使用酸酚法测量的在BSA中的糖水平为0.7%(表1)。
实施例1.在具有HW-55树脂的柱上从商购制剂样品中分离BSA单体的方法
用60mg商购BSA装载柱(1.6×100cm)的顶部，所述柱装载有用0.1M、pH5.65的含1%正丁醇的乙酸盐缓冲剂平衡的HW-55S树脂(Toyopearl,Tosh Bioscience)。12%SDS/PAGE分析确认了商购试剂的多分散性：除了分子量为69kDa的单体白蛋白之外，我们观察到还存在Mw为200kDa的聚集体以及更低分子量的衍生物(图2，泳道2)。在室温下以1.2ml/10min的速率用平衡缓冲剂从柱中洗脱蛋白。在峰D中的从柱中洗脱的级份中(图1A)，存在电泳迁移率对应于白蛋白(分子质量69kDa)的均质蛋白，这是使用在12%SDS/PAGE中的电泳分析而证实的(图2，泳道5)。使用新的方法，通过在Zeta-seizer NanoZS分析仪(Malvern,GB)中使用动态光散射(DLS)测定蛋白分子的直径，将分离的蛋白鉴定为白蛋白。在从HW-55S柱中收集的级份53-58中观察到的分子直径为7nm(图3，具有黑色正方形的实线)，其对应于单体BSA的文献数据[39]。
在所获得的级份中的总糖含量的酚法分析证实不存在糖污染物(表1)。
实施例2.在Sephadex G-200葡聚糖凝胶(Amersham Pharmacia)中使用分子筛分离从商购制剂样品中分离BSA单体的方法
将50mg的商购BSA样品装载至用pH7.5的含0.02%NaN3的PBS(磷酸盐缓冲盐水)平衡的Sephadex G-200柱的顶部上。在室温下以1.2ml/15min的速率用平衡缓冲剂进行蛋白洗脱。在三个蛋白峰中洗脱的白蛋白级份的事实证明商购BSA的非同质性(图1B)。SDS/PAGE分析证明在峰C中发现BSA(图2，泳道8)。其构成了装载的商购制剂的质量的60%。使用酚法检测的糖含量为约1%的糖，并且在所得单体蛋白中，其略高于商购样品(表1)，这表明这是葡聚糖污染物的结果。这证明了通过Sephadex G-200凝胶过滤来获得纯BSA单体的不适用性。
实施例3.在DEAE-Sephadex A-50阴离子凝胶(Amersham Pharmacia)上使用离子交换色谱法从商购制剂中分离BSA单体的方法
用溶解于1ml平衡缓冲剂中的600mg商购BSA来装载柱(3×25cm)，所述柱装有阴离子凝胶DEAE-Sephadex A-50且用0.02M、pH8.0的磷酸盐缓冲剂平衡。用0.02-0.3M的pH8.0的磷酸盐的浓度梯度来洗脱与离子交换剂混合的白蛋白。在峰A(图1C)中的较低范围的洗脱缓冲剂浓度下以1ml/20min的速率洗脱的蛋白在电泳方面为均质的(图2，泳道9)。12%SDS/PAGE分析表明在峰C中以更高磷酸盐梯度浓度洗脱的商购蛋白样品的高分子量聚集体(图2，泳道11)。可能由于碳水化合物对蛋白的非酶修饰(糖化)，形成的蛋白衍生物具有更高的负电荷(可用的碱性氨基酸残基被糖化剂封闭)并与阴离子凝胶结合得更牢固。
使用酚法测定的峰A中收集的级份中的总糖含量为0.9%(表1)。为此，使用诸如DEAE-Sephadex的阴离子交换剂不适于获得纯BSA单体。
实施例4.在使用BSA单体与宋内志贺菌(Shigella sonnei)PhII的脂多糖的核心寡糖形成糖轭合物的实例中显示出本发明的显著性
在37℃温度下，商购BSA样品和单体白蛋白(采用在HW-55S上进行分级而获得的)用细菌寡糖(蛋白与寡糖的摩尔比为1:200)孵育16小时。基于早期报道[22,23]来选择修饰条件，并且在12%分离凝胶上通过SDS/PAGE电泳分析反应产物。在用寡糖孵育的BSA单体样品中，我们观察到糖轭合物，而未用寡糖的对照样品不产生这样的产物(图4，分别为泳道5和4)。由于商购BSA制剂的修饰，我们获得了Mw为130-530kDa的糖轭合物，在图4的泳道2中可见。在未用寡糖处理的对照商购样品中观察到类似组合物，尽管其较不强烈(图4，泳道3)。因此，使用商购BSA作为评价蛋白修饰的模型蛋白由于其在结果中引起大量偏差而应被排除。仅纯BSA使获得正确的结果成为可能；为此，优选使用HW-55S树脂从商购制剂中分离BSA。
表1.BSA样品中总糖含量
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Claims (6)
1.在12%分离凝胶中通过SDS/PAGE用电泳测定的分析纯的单体牛血清白蛋白。
2.如权利要求1所述的分析纯的单体牛血清白蛋白，其特征在于BSA分子的直径基本为7nm。
3.获得单体血清白蛋白的方法，其特征在于所述方法包括：
a)BSA制剂的样品的制备；
b)使用平衡缓冲剂在用缓冲剂平衡的树脂上进行的阶段a)中获得的样品的纯化色谱法，所述树脂优选为HW55，所述缓冲剂优选为0.1M的pH5.65的含1%正丁醇的乙酸盐缓冲液。
4.如权利要求2所述的方法，其特征在于阶段a)包括除去疏水性配体，优选地将所述样品通过炭来除去所述疏水性配体。
5.如权利要求2或3所述的方法，其特征在于在阶段b)中的所述纯化在室温下以1.2ml/10min的速率实施。
6.鉴定白蛋白的方法，其特征在于所述方法包括使用动态光散射测定在使用权利要求1所述的方法获得的级份中蛋白分子的直径，其中携带单体BSA的级份为具有直径基本为7nm的分子的级份。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107228910A (zh) * | 2017-01-24 | 2017-10-03 | 浙江海隆生物科技有限公司 | 一种油乳剂蛋白亚单位疫苗中蛋白含量和纯度的检测方法及其应用 |
| CN107228910B (zh) * | 2017-01-24 | 2018-08-24 | 浙江海隆生物科技有限公司 | 一种油乳剂蛋白亚单位疫苗中蛋白含量和纯度的检测方法及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| PL221899B1 (pl) | 2016-06-30 |
| US20140205839A1 (en) | 2014-07-24 |
| AU2012320205A1 (en) | 2013-11-21 |
| PL394784A1 (pl) | 2012-11-19 |
| WO2013050830A1 (en) | 2013-04-11 |
| IL229219A0 (en) | 2014-01-30 |
| EP2705048B1 (en) | 2019-07-17 |
| EP2705048A1 (en) | 2014-03-12 |
| CA2835074A1 (en) | 2013-04-11 |
| US9074017B2 (en) | 2015-07-07 |
| JP2014514343A (ja) | 2014-06-19 |
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