化学交联制备蛋白质二聚体
文献类型：学位论文
| 作者 | 胡涛 |
| 学位类别 | 博士 |
| 答辩日期 | 2002 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 中国科学院过程工程研究所 |
| 导师 | 苏志国 |
| 关键词 | N－经基唬拍酰亚胺一（间马来酰亚胺基）苯甲酸酷 N－琉拍酞亚胺一3（2一二硫毗陡）丙酸醋 固相吸附法 化学偶联法 蛋白二聚体 |
| 其他题名 | Preparation of Protein Dimer by Chemical Crosslinking |
| 学位专业 | 生物化工 |
| 中文摘要 | 用化学方法对蛋白质进行改造是现代生物技术发展的一个重要方向。其中，将两个蛋白质分子用化学交联技术连接在一起具有重要的应用价值。本文首先考察同型双功能试剂在溶液中对两个蛋白质分子的交联。采用同型双功能试剂戊二醛对牛血清白蛋白（BSA）和牛血红蛋白（BHb）进行交联，得到了有二聚体、三聚体和多聚体的混合物，其中目标产物BSA一BHb占19％。用阴离子交换色谱可以分离交联产物，得到BSA一BHb交联物。该交联物的PS。为2 1 ．6 mm Hg，Hill系数为2．12。与天然血红蛋白相比，Bohr系数下降为37％，氯离子效应下降了43％。接下来考察了带有不同反应基团的异型双功能试剂在液相中对两个蛋白质的交联。选用N－轻基琉拍酞亚胺一（间马来酰亚胺基）苯甲酸醋（MBS）对牛血清白蛋白（B SA）和牛血红蛋白（B Hb）进行交联。预先封闭了BsA上可能存在的琉基，让MBs的轻基墟拍酰亚胺酷基团与BSA上的氨基反应，间一马来酞亚胺基团与BHb的p一93半肤氨酸的琉基反应，从而形成了BSA一BHb交联物，避免了BHb一BHb、BSA一BSA的形成，偶联率达到97％。用DEAE sepharose Fast Flow阴离子交换柱纯化偶联物，去除未反应的BHb，纯化收率为62％，偶联物的分子量约为1 27 kD。，偶联物的PS。为27．4～Hg，Hin系数为2．03。本文还用另一个异型双功能试剂N－唬拍酞亚胺一3（2一二硫毗咤）丙酸醋为交联剂，交联了牛血清白蛋白（B SA）和过氧化氢酶（CAT）偶联物，得到了BSA一CAT的双聚体，偶联物的分子量约为370kD。。交联后CAT、抗蛋白水解酶降解的能力、热稳定性和抗脉变性能力都显著提高。虽然异型双功能试剂能够获得更高的产物收率，但其昂贵的价格必然造成大规模生产的成本过高。为此本文对同型双功能试剂进行了下列探索（1）用两个不同的同型双功能试剂进行双交联反应。以两个血红蛋白的二聚体为例，先采用第一个交联剂二甲基己二亚胺酸酷（DMA）与血红蛋白反应，使交联反应发生在血红蛋白分子的内部，形成分子内交联以减少蛋白质表面的自由氨基数量，然后加入第二个交联剂戊二醛。利用血红蛋白表面剩余的氨基进行两个分子间的交联·获得了分子量分布较窄的交联产物，其中分子量为128 kDa的二聚体占多数。血红蛋白二聚体的P50值为15.1～Hg，Hill系数为1．68，氧传递效率11．8％。（2）用固相吸附法辅助同型双功能试剂反应，制备分子量分布均匀的交联产物。本文共进行了两种交联产物的制备。首先是BSA一BHb的制备，将牛血清白蛋白吸附到QSePharoseFastFlow介质上，随后与戊二醛反应。牛血清白蛋白在介质上的空间距离限制了牛血清白蛋白分子的自身聚合。因此，产物大多为戊二醛活化的单体牛血清白蛋白。用SuPerdex 200凝胶过滤柱分离活化的单体牛血清白蛋白，与牛血红蛋白在4oC和pH 9 ．5的条件下进行偶联反应，反应时间为8小时，产率为64％。在脱氧条件下制备的偶联物的P50值分别为19．1 mm Hg，Hill系数为1.96，氧传递效率12．9％。其次是BHb一BHb二聚体的制备，将血红蛋白吸附在Q Sepharose Fast Flow层析介质上，随后与戊二醛反应。由于有些血红蛋白分子在层析介质上的距离较近，与戊二醛反应时可生成血红蛋白二聚体。在脱氧条件下制备的血红蛋白二聚体的PS。值为巧．9～Hg，氧传递效率为14．2％，Hin系数为1．62。 |
| 英文摘要 | Chemical modification of protein is one of the important research directions for the development of modern biotechnology, in which two proteins conjugation using chemical crosslinking method is of applied significance. First, two proteins conjugation by homobifunctional reagent was investigated in aqueous solution. Glutaraldehyde was used to couple bovine serum albumin (BSA) and bovine hemoglobin (BHb). The mixtures containing dimers, timers and polymers were obtained, in which the ratio of BSA-BHb conjugate is 19%. Anion-exchange chromatography was used to separate the BSA-BHb conjugate. P5o and the Hill coefficient for the conjugate were 21.6 mm Hg and 2.12, respectively. By comparison with native hemoglobin, Bohr coefficient and chloride ion effect of the conjugate decreased to 37% and 43%, respectively. Second, two proteins conjugation by heterobifunctional reagent was investigated in aqueous solution. BSA-BHb conjugate was prepared using 3-maleimidobenzoic acid iV-hydroxysuccinimide ester. iV-hydroxysuccinimide ester group was used to react with the amino group on BSA, followed by reaction between 3-maleimidobenzoic acid group and SH- group of cysteine on (3-93 of BHb, The conjugate with the yield of 97% was purified on DEAE Sepharose Fast Flow chromatography. The conjugate was confirmed with molecular weight of 137 kDa. P50 of the conjugate was 27.4 mm Hg, while the Hill coefficient was 2.03. In addition, iV-succinimidyl 3-(2-pyridyldithio) propionate was used to prepare a bovine serum albumin-catalase conjugate. The molecular weight of the conjugate is 370 kDa. Heat stability, and the ability to resist protease and urea denaturation of the conjugate increased significantly. Though high product yield can be obtained by heterobifunctional reagent, its highprice will limit the large-scale production. Thus, homobifunctional reagent was investigated in details: (1) Double cross-linkage method was proposed to prepare the dimeric hemoglobin trtramers. DMA was used to block the amino groups of BHb, followed by reaction with glutaraldehyde. The cross-linking product with narrow molecular weight distribution (mainly 128 kDa) was thus obtained. P50, the Hill coefficient and the oxygen transporting efficiency for the dimeric hemoglobin trtramers were 15.1 mm Hg, 1.68 and 11.8%, respectively. (2) Homobifunctional reagent in combination with solid phase adsorption method was proposed to prepare well-defined cross-linking product. First, a bovine serum albumin-bovine hemoglobin conjugate was prepared. After adsorption by Q Sepharose Fast Flow, BSA molecules were allowed to react with glutaraldehyde. The spacing out of BSA molecules on the solid phase was assumed to limit BSA polymerization, except some molecules bound closely resulting in minor dimers formation. Following the elution procedure, the monomeric BSA was separated by Superdex 200 gel filtration and then reacted with bovine Hb at 4°C and pH 9.5. P50, the Hill coefficient and the oxygen transporting efficiency for the conjugate were 15.9 mm Hg, 1.62 and 14.2%, respectively. Second, the dimeric hemoglobin trtramers was prepared. BHb was adsorbed by Q Sepharose Fast Flow media, followed by reaction with glutaraldehyde and elution procedure. Some BHb molecules were adsorbed with a short intermolecular distance on the solid phase, thereby resulting in formation of dimeric BHb tetramers. The reaction under anaerobic conditions provided the dimeric BHb tetramers with P50 value of 15.9 mm Hg, oxygen transporting efficiency of 14.2% and the Hill coefficient of 1.62. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 143 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1351]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 胡涛. 化学交联制备蛋白质二聚体[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2002. |
入库方式： OAI收割
来源：过程工程研究所
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