CN1622760A - 提取、纯化和酶促修饰用作降低血胆固醇剂的大豆7S球蛋白α’亚基的方法 - Google Patents
提取、纯化和酶促修饰用作降低血胆固醇剂的大豆7S球蛋白α’亚基的方法 Download PDFInfo
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Abstract
提取、纯化和酶促修饰β－伴大豆球蛋白α’亚基的方法，其中β－伴大豆球蛋白选择性提取自磨碎的脱脂大豆，然后用含水乙醇处理而沉淀；接下来对富集的级分在变性条件下进行金属亲和层析(MAC)以获得α’亚基，该α’亚基用胰凝乳蛋白酶处理，然后进行进一步的亲和层析步骤以重新得到该多肽的氨基端区域(MW 28,000Da)。
Description
本发明涉及提取、纯化和酶促修饰β-伴大豆球蛋白(β-conglycinin)α’亚基的方法。
根据本发明，β-伴大豆球蛋白选择性提取自磨碎的脱脂大豆，然后用含水乙醇处理而沉淀；接下来对富集的级分在变性条件下进行金属亲和层析(MAC)以获得α’亚基。α’亚基用胰凝乳蛋白酶处理，然后进行进一步的亲和层析步骤以重新得到该多肽的氨基端区域(MW 28,000Da)。
技术背景
大豆及其衍生物已知的胆固醇降低特性与异黄酮类的含量有关(Kirk等人，1998)并与蛋白质的含量有关(Anderson等人，1995)。
大豆蛋白质主要由大豆球蛋白(11S级分)和β-伴大豆球蛋白(7S级分)组成，后者由α、α’和β三个亚基组成(Thanh和Shibasaki，1976)。对大豆蛋白质进行的研究表明7S级分(Lovati等人，1992，1996)，尤其是α’亚基(Manzoni等人，1998)能激活LDL受体，因此主要负责降低胆固醇血浆水平。事实上，用7S球蛋白处理肝细胞系造成α和α’亚基的彻底降解和对LDL受体活性的刺激，然而β亚基没有被降解，因此受体也没有激活。此外，7S级分缺失α’亚基的大豆突变体不能改变受体活性，甚至在高浓度下也不能改变受体活性。
基于这些实验观察的结果，需要一些方法用于获得纯化的β-伴大豆球蛋白、以及回收和纯化α’亚基，由后者可通过酶促处理接下来可获得特异的氨基酸序列，而不是通过多肽合成获得的。
Than等人(1975和1976)提出，后来经O′Keefe等人(1991)改进的一个方法是用来根据大豆球蛋白和β-伴大豆球蛋白在不同pH值下不同的溶解度来分离它们；但是交叉污染还是很严重，而且需要凝胶过滤和亲和层析，这些方法花费昂贵，在工业规模下实施很困难。Nagano等人(1992)改进的方法尽管允许提高级分的纯度，但还是一种花费昂贵只适用于实验室规模的方法。
最近，Wu等人(1999)描述了一种在半工业试验规模下分离大豆球蛋白和伴大豆球蛋白的方法。大豆球蛋白经连续两次水提取，pH为8.5，接着将上清液用0.98g/l的亚硫酸氢盐溶液处理而沉淀，同时，向来自大豆球蛋白沉淀的母液中加入0.25M NaCl，将pH值调到4.8而沉淀伴大豆球蛋白。这种方法用来处理大量的起始材料，也提供高蛋白质产量，但是级分的纯度仍然不理想；特别的是β-伴大豆球蛋白被降解，显然是在用水渗滤期间发生的，渗滤是减少过量亚硫酸氢盐离子并去除盐的必要处理。
上述方法不仅没有产出纯的β-伴大豆球蛋白，而且最重要的是没有考虑提取和纯化α’亚基。
根据本发明，按照常规方法通过在水介质中从磨碎的脱脂大豆提取来制备β-伴大豆球蛋白中富含的固体级分，接下来上清液用含水乙醇沉淀；所得的级分然后在变性条件下通过金属亲和层析(MAC)纯化，以产生纯的α’亚基，α’亚基用胰凝乳蛋白酶处理以得到氨基端区域，它显然具有最高的LDL受体活化活性。
发明详述
本发明涉及用于选择性提取、纯化和酶促修饰大豆的β-伴大豆球蛋白α’亚基的方法，该方法包括以下步骤：
a)用亚硫酸氢钠水溶液在弱酸性pH下从磨碎的脱脂大豆中提取得到β-伴大豆球蛋白富集的可溶性蛋白质级分；
b)用乙醇处理而沉淀来自步骤a)的β-伴大豆球蛋白级分；
c)在变性剂条件下通过金属亲和层析(MAC)纯化步骤b)得到的沉淀级分，以分离α’亚基；
d)用蛋白水解酶酶促处理来自步骤c)的α’亚基，并进一步通过MAC层析纯化；
e)用有机溶剂沉淀α’亚基。
β-伴大豆球蛋白的富集在图1中显示。起始材料是通过用溶剂去除脂级分而脱脂的大豆粉。在弱酸性pH下用亚硫酸氢钠水溶液提取该起始材料。使用14-16倍起始材料重量的溶液体积，优选14.5-15.5倍。亚硫酸氢盐的浓度为0.80-1.20g/l，优选地为0.90-1.10g/l，最优选地为0.95-1.05g/l。提取在温度-2-8℃进行14到18小时。根据本发明一个优选的实施方案，提取是在0-4℃，pH值为6.4的0.98g/l 15倍体积亚硫酸氢盐溶液中进行16小时。
在上述pH值和温度条件下，大豆球蛋白的溶解度很低，因此它们和其它不可溶物质一起沉淀下来。沉淀物通过离心分离，将可溶的级分在20℃-30℃的温度，优选地在室温25℃，用35-60％(体积/体积)含水乙醇处理，优选地为40％含水乙醇。将上清液离心去掉，并将主要由β-伴大豆球蛋白组成的沉淀物冷冻干燥。得到的粉末进行下一步(图2)。
用金属亲和层析的方法(Ostrove和Weiss，1990)来分离和纯化α’亚基的选择取决于该亚基与金属离子，如Zn2+和Ni2+的结合能力，因为该亚基比起α亚基和β亚基来组氨酸含量更高(Thanh和Shibasaki，1978)。
使用与锌或镍，优选地使用与锌结合的基质。根据本发明一个优选的实施方案，此基质由亚氨基二乙酸琼脂糖组成。将冷冻干燥的蛋白质在由50mM Tris、0.5M NaCl、pH7.2组成的、含5-8M尿素，优选地含5M尿素的变性缓冲液中悬浮。在这些条件下，α’亚基选择性地结合到基质上，α亚基和β亚基可以通过用上述缓冲液洗脱而去除；α’亚基随后用在相同缓冲液或蒸馏水中含0.1M咪唑的溶液洗脱。
将富集α’亚基的蛋白质级分收集起来并用有机溶剂处理，使蛋白质沉淀，优选地用冷丙酮处理。所使用的丙酮体积为级分体积的2-5倍，优选地3-4倍，在-10--30℃，优选地在-15--25℃之间的温度。根据本发明一个优选的实施方案，应用-20℃，3倍体积的丙酮。所得的沉淀物用离心法分离，重悬在乙醇中，优选95％的乙醇，然后进一步离心、冷冻干燥。冷冻干燥物包括94％的蛋白质，其α’亚基富集量是起始材料的十倍。
表1显示了从大豆粉提取出来的β-伴大豆球蛋白和α’亚基的产量。
表1
| 蛋白质级分 | 起始材料 | 提取产量(％重量) |
| β-伴大豆球蛋白 | 脱脂粉 | 18.7 |
| α’亚基 | β-伴大豆球蛋白 | 11.0 |
| α’亚基 | 脱脂粉 | 2.1 |
α’亚基的多肽片段是将来自前面步骤的冷冻干燥物用蛋白水解酶进行酶促处理制备得到的。根据本发明一个优选的实施方案，蛋白水解酶是胰凝乳蛋白酶，且所得到的片段主要由分子量28,000Da的氨基端区域组成。
流程如下所述：从前面几步得到的冷冻干燥物以5mg/ml溶解于pH值7.5-8.5，含有1.6M尿素的0.2M NH4HCO3中。将胰凝乳蛋白酶以1∶10-1∶50比例，优选地1∶25w/w加入上述底物中，在37℃搅拌温育24小时。接下来实施前面所述的金属亲和层析这一步骤。
用咪唑洗脱下来的物质包含三个多肽片段，主要的一个具有分子量28000Da，它组成α’亚基的N-末端区域。
将α’亚基和胰凝乳蛋白酶片段施与大鼠(表2)证明两者都显著降低了胆固醇和总甘油三酯血浆水平。特别是胰凝乳蛋白酶片段被证明在降低胆固醇水平方面它不仅比其他大豆成分更有效，而且比安妥明更有效，并且它还提供了不相上下的对甘油三酯的结果。
该生物学实验的结果表明了根据本发明的方法获取的产品，特别是α’亚基及其片段可以用来作为药物，特别是用于治疗那些需要降低胆固醇和/或甘油三酯血浆水平的病症。所述化合物可以单独使用或与其它活性成分组合，并与合适的载体混和，用来制备药物组合物，特别是用于治疗高脂血症的药物组合物。此外，它们还可以用来在上面提到的条件下制备用于膳食疗法的补充物或食品。
实施例
第一步：从大豆纯化7S球蛋白
起始材料是磨碎的大豆，根据索格利特法脱脂，使用戊烷做溶剂。
蛋白质用0.98g/l的亚硫酸氢钠溶液以15倍脱脂磨碎的大豆体积提取，在温度0-4℃提取16小时，保持pH值为6.4。离心后，上清液用40％(体积/体积)乙醇溶液在室温下处理。将得到的富集β-伴大豆球蛋白和含有起始材料双倍浓度的α’亚基的沉淀物冷冻干燥。
第二步：α’亚基的纯化
β-伴大豆球蛋白富集级分在变性缓冲液(50mM Tris、0.5M氯化钠、pH值为7.2)中重悬，缓冲液中含5M尿素，并在与锌结合的亚氨基二乙酸琼脂糖基质(Sigma)上进行金属亲和层析纯化。未结合的蛋白质物质用上述相同的缓冲液洗脱，结合的蛋白质物质主要由α’亚基组成，被含0.1M咪唑的相同缓冲液或蒸馏水洗脱。
将富集α’亚基的级分用3-4倍体积的-20℃丙酮处理；所得的沉淀物室温下在40％乙醇中悬浮，然后离心、冷冻干燥。所得到的粉末包含94％的蛋白质，并且α’亚基富集量是起始材料的十倍。
第三步：α’亚基的酶促处理
从前面几步得到的冷冻干燥物以5mg/ml的浓度在pH值7.5-8.5，含有1.6M尿素的0.2M NH4HCO3中溶解。将胰凝乳蛋白酶以1∶25w/w比例加入蛋白质底物中，温度37℃下搅动温育24小时，然后进行上述的金属亲和层析纯化。被树脂滞留并用0.1M咪唑洗脱下来的物质包含三个多肽片段，主要的一个具有分子量28000Da，它由α’亚基的N-末端区域组成。
生物学实验
动物
使用雄性大鼠CD SPF/VAF，重量为75-100g。这些动物饲养在聚碳酸酯笼子里(每个笼子4-5只动物)，笼子放在自动控制光线(12小时明/12小时暗的循环)、温度(21±1℃)、湿度(60±5％)的环境里。
实验方法
饲养七天后，将这些动物随机分成七组，每组20只大鼠(表2)。28天期间之内，一组喂养正常饮食(cod.014RF25C；Mucedola S.r.l.，SettimoMilanese，MI，Italy)，而其他组喂养由1％胆固醇、0.5％胆酸和25％氢化椰子油组成的高胆固醇饮食(批号为332000，2000.09.01制备；LaboratorioDottori Piccioni，Gessate，MI，Italy)，自由获取水。该饮食每天(40g，上午9:00)喂养它们，剩下没吃的称重。按照下面进行处理。
组1(对照)：给动物喂养正常饮食，用0.5％的羧甲基纤维素溶液口服处理28天。
组2：给动物喂养高胆固醇饮食，用0.5％的羧甲基纤维素溶液口服处理28天。
组3：给动物喂养高胆固醇饮食，用200mg/kg剂量的安妥明口服处理28天。
组4：给动物喂养高胆固醇饮食，用200mg/kg剂量的大豆总蛋白质提取物口服处理28天。
组5：给动物喂养高胆固醇饮食，用50mg/kg剂量的β-伴大豆球蛋白口服处理28天。
组6：给动物喂养高胆固醇饮食，用10mg/kg剂量的α’亚基口服处理28天。
组7：给动物喂养高胆固醇饮食，用1mg/kg剂量的α’亚基胰凝乳蛋白酶片段口服处理28天。
总胆固醇和甘油三酯血浆水平在28天的处理和16小时的禁食后进行检测。将动物用乙醚麻醉，使血液从下腔静脉流入含有EDTA(1mg/ml)的试管中。在4℃，3000转/分离心15分钟后，血浆回收、冷冻，并储存在-20℃温度，直到检测为止。
总胆固醇和甘油三酯血浆浓度(表2中报道)由常规的酶促鉴定法来确定。
表2
| 处理 | 总胆固醇(mg/dL) | 总甘油三酯(mg/dL) |
| 组1 | 55.4±3 | 105.1±7.2 |
| 组2 | 284.1±10.3 | 226.9±12.6 |
| 组3 | 191.2±8.0 | 139.1±5.8 |
| 组4 | 236.1±10.2 | 176.9±8.1 |
| 组5 | 196.4±7.6 | 146.7±5.9 |
| 组6 | 182.2±12.1 | 150.1±9.8 |
| 组7 | 175.8±7.9 | 140.3±7.4 |
参考文献
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Claims (19)
1.用于选择性提取、纯化和酶促修饰大豆的β-伴大豆球蛋白α′亚基的方法，该方法包括下列步骤：
a)用亚硫酸氢钠水溶液在弱酸性pH下提取磨碎的脱脂大豆，以获得β-伴大豆球蛋白富集的可溶性蛋白质级分；
b)通过用乙醇处理而沉淀来自步骤a)的β-伴大豆球蛋白级分；
c)在变性剂条件下通过金属亲和层析(MAC)纯化来自步骤b)的沉淀级分，以分离α’亚基；
d)用蛋白水解酶酶促处理来自步骤c)的α’亚基，并进一步通过MAC层析纯化；
e)用有机溶剂沉淀α’亚基。
2.如权利要求1所述的方法，其中提取是用15倍体积的0.98g/l亚硫酸氢钠溶液于pH6.4进行。
3.如权利要求1所述的方法，其中步骤b)的沉淀用40％乙醇进行。
4.如权利要求1所述的方法，其中步骤c)中的MAC在连接有锌或镍的基质上进行。
5.如权利要求4所述的方法，其中基质上连接有锌。
6.如权利要求4或5所述的方法，其中基质由琼脂糖亚氨基二乙酸组成。
7.如权利要求1所述的方法，其中步骤c)的MAC中所用的变性剂是尿素。
8.如权利要求1所述的方法，其中步骤d)的酶促处理所用的蛋白水解酶是胰凝乳蛋白酶。
9.如权利要求1所述的方法，其中步骤e)中用于α’亚基的沉淀溶剂是丙酮。
10.如权利要求1所述的方法，其中β-伴大豆球蛋白富集级分和α’亚基通过冷冻干燥而稳定。
11.来自大豆的β-伴大豆球蛋白富集级分和α’亚基，可由权利要求1-10所述的方法获得。
12.大豆β-伴大豆球蛋白α’亚基的多肽片段，其可由权利要求1-10所述的方法获得。
13.氨基端多肽片段，其可根据权利要求8所述的方法得到。
14.用作药物的权利要求12的大豆β-伴大豆球蛋白α’亚基的多肽片段。
15.用作药物的权利要求12的氨基端多肽片段。
16.权利要求12的大豆β-伴大豆球蛋白α’亚基的多肽片段的用途，用来制备治疗高脂血症的药物。
17.权利要求12的多肽片段的用途，用来制备治疗高脂血症的药物。
18.药物组合物，其含有与合适载体混和的、单独或与其他活性成分组合的权利要求12或13的多肽片段。
19.含有权利要求12或13的多肽片段的补充物或食品。
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| CN102753571A (zh) * | 2009-06-17 | 2012-10-24 | 因德纳有限公司 | 参与HEP G2细胞中胆固醇内稳态的大豆7S球蛋白α’亚基延伸区的克隆、酵母表达、纯化和生物学活性 |
| CN114874301A (zh) * | 2022-05-10 | 2022-08-09 | 吉林农业大学 | 不同亚基组成β-伴大豆球蛋白的制备方法及应用 |
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| JP4687463B2 (ja) * | 2003-12-11 | 2011-05-25 | 不二製油株式会社 | 改良大豆７ｓたん白及びその製造法 |
| JP5382840B2 (ja) * | 2005-08-23 | 2014-01-08 | 特定非営利活動法人プライメイト・アゴラ | サルモデルで薬効が確認された骨粗鬆症の予防又は治療剤 |
| EP2264054A1 (en) * | 2009-06-17 | 2010-12-22 | Indena S.p.A. | Cloning, yeast expression, purification and biological activity of a truncated form of the soybean 7S globulin alfa ' subunit involved in Hep G2 cell cholesterol homeostasis |
| CN101904406B (zh) * | 2010-07-02 | 2012-07-25 | 山西大学 | 一种葵花籽多肽的制备方法和用途 |
| CN104163767B (zh) * | 2014-07-25 | 2017-01-18 | 湖北泓肽生物科技有限公司 | 一种氨基酸和多肽水溶性物质的纯化方法 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102387711A (zh) * | 2008-12-31 | 2012-03-21 | 索莱有限责任公司 | 蛋白质水解产物组合物 |
| CN102753571A (zh) * | 2009-06-17 | 2012-10-24 | 因德纳有限公司 | 参与HEP G2细胞中胆固醇内稳态的大豆7S球蛋白α’亚基延伸区的克隆、酵母表达、纯化和生物学活性 |
| CN102753571B (zh) * | 2009-06-17 | 2015-11-25 | 因德纳有限公司 | 参与HEP G2细胞中胆固醇内稳态的大豆7S球蛋白α’亚基延伸区的克隆、酵母表达、纯化和生物学活性 |
| CN114874301A (zh) * | 2022-05-10 | 2022-08-09 | 吉林农业大学 | 不同亚基组成β-伴大豆球蛋白的制备方法及应用 |
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