Developement of the Method for the Membrane Proteins Crystalization and Its Application to Ecoli Membrane bound Phospholipase A

膜蛋白结晶方法的建立及其在大肠杆菌膜结合磷脂酶A中的应用

• 批准号: 04671331
• 负责人: SETAKA Morio
• 金额: $ 1.28万
• 依托单位: TEIKYO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04671331/
• 关键词: Crystalization of membrane proteins; Membrane bound phospholipase A; Two dimensional crystals; Antibody column of membrane proteins; Electrophoresis purification of proteins; 蛋白の電気泳動精製

We planned to develope the method for crystalization of membrane bound proteins. The method consists of two steps, firstly formation of two dimensional crystal of the protein utilizing the surface of water and then accumulation of it.In order to perform this experiment, a large amount of the membrane protein is necessary. So we decided to purify E.coli membrane bound, detergent-resistant phospholipase A (DRPLA) since this enzyme had been well characterized for gene manipulation of DRPLA,generating an overproducing E.coli strain.As the first step of purification, acetone precipitate of cell homogenate was solubilized with SDS and pretreated with Sepharose Q before Sepharose Q column chromatography. The crude preparation after the first column chromatography was indicated by SDS PAGE to still contain impurity proteins. As the next step, we performed an affinity column-chromatography of anti DRPLA polyclonal antibody because we failed in obtaining monoclonal antibody against DRPLA.It was … More found that DRPLA solubilized by detergents such as triton x-100, tween 20, SDS was unable to bind to antibodies coupled to column resins, perhaps because DRPLA would be entrapped into micelles of detergents deeply. In stead of detergent solution, DRPLA was found in the partial mixture of aqueous solution with orgnic solvents to bind to antibodies, but DRPLA fractions eluted from the DRPLA-binding antibody columns still contained some impurities and, in addition, recovery yield of DRPLA was very low. Then, we attempted SDS polyacrylamide gel electrophoresis purification of DRPLA.A large scale of preparative electrophoresis was performed with the crude preparation and DRPLA band (29KD) was cut out from SDS PAGE gel. The apparatus was constructed for electrical elution of DRPLA from DRPLA band gel pieces. Some preliminary experiments indicated that almost pure DRPLA was eluted quite effectively from gel pieces and that this electrophoresis method is very useful for purification of other proteins. We shall continue this project with the obtained pure DRPLA. Less

我们计划开发膜结合蛋白的结晶方法。该方法包括两个步骤，首先利用水表面形成蛋白质的二维晶体，然后进行积累，为了进行这一实验，需要大量的膜蛋白质。因此，我们决定纯化与膜结合的、耐洗涤剂的磷脂酶A(DRLA)，因为这种酶已经很好地用于DRLA的基因操作，产生了一株高产的大肠杆菌菌株。作为纯化的第一步，细胞匀浆中的丙酮沉淀用十二烷基硫酸钠溶解，并在SephoseQ柱层析之前用SephoseQ进行预处理。第一柱层析后的粗品经SDS-PAGE分析表明仍含有杂质蛋白。下一步，由于未能获得抗DRPLA的单抗，我们进行了抗DRPLA多克隆抗体的亲和柱层析，这是…进一步研究发现，Triton x-100、吐温20、十二烷基硫酸钠等洗涤剂增溶的DRPLA不能与柱树脂偶联的抗体结合，这可能是因为DRPLA会被深深地包裹在洗涤剂胶束中。在与抗体结合的水溶液和有机溶剂的部分混合物中发现了DRPLA而不是洗涤剂溶液，但从DRPLA结合的抗体柱中洗脱出来的DRLA组分中仍然含有一些杂质，而且DRLA的回收率很低。在此基础上，我们尝试了用SDS-聚丙烯酰胺凝胶电泳法纯化DRPLA，并用粗品进行了大规模的制备电泳法，从SDS-PAGE凝胶中切出了一条29KD的DRLA条带。该装置用于从DRPLA条带凝胶片中电洗脱DRPLA。初步实验表明，从凝胶碎片中可以相当有效地洗脱出几乎纯净的DRLA，这种电泳法对其他蛋白质的纯化也非常有用。我们将继续这个项目，获得纯净的DRPLA。较少