谷胱甘肽亲和吸附介质的制备及其对谷胱甘肽硫转移酶的纯化
文献类型：学位论文
| 作者 | 张海龙 |
| 学位类别 | 硕士 |
| 答辩日期 | 2007-06-03 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 过程工程研究所 |
| 导师 | 苏志国 |
| 关键词 | 亲和层析介质 环氧活化 配基偶联 谷胱甘肽S转移酶 纯化 |
| 其他题名 | Preparation of Glutathione Affinity Adsorbents and Purification of Glutathione S-Tansferase (GST) |
| 学位专业 | 生物化工 |
| 中文摘要 | 谷胱甘肽亲和吸附介质应用广泛，可用于分离纯化不同来源的谷胱甘肽S－转移酶及以谷胱甘肽S－转移酶为标签的重组融合蛋白。本文以谷胱甘肽为配基，琼脂糖微球为骨架，探索研究了谷胱甘肽配基共价偶联到琼脂糖微球骨架上的合成工艺条件。 首先，以1, 4-丁二醇-二缩水甘油醚（BDGE）为活化剂，对琼脂糖凝胶上的羟基进行活化，考察了BDGE加入量、反应时间、温度、NaOH溶液的初始浓度以及NaBH4加入量等因素对介质上生成的环氧基密度的影响，确立了琼脂糖基质环氧活化的优化条件；其次，以还原型谷胱甘肽作为配基与琼脂糖凝胶上的环氧基进行配基偶联反应，采用正交实验方法考察了谷胱甘肽加入量、缓冲液的pH值、反应温度对介质配基密度的影响，获得了合适的偶联条件。最后，确定了通过反应时间来控制配基密度的方法并比较了不同双环氧试剂对环氧基密度及配基密度的影响。 在BDGE活化的基础上，本文又探索了一种新的活化方法—烯丙基缩水甘油醚（AGE）活化法。利用AGE活化琼脂糖凝胶的羟基，在凝胶上引入了相对惰性的双键基团；然后滴加溴水使其转化为邻溴醇，再在碱性条件下脱溴化氢形成环氧基。该法解决了BDGE活化时形成时的环氧基水解导致环氧基减少、难以获得高配基密度的难题。通过研究活化过程中的烯丙基转化为环氧基的各种影响因素，获得了环氧化密度可控的条件，使偶联后的配基密度从原来的281μmol/g-dry gel(BDGE)提高到660μmol/g-dry gel(AGE)。 对研制的介质进行理化性质的考察，结果表明该介质具有良好的机械和化学稳定性，可以用6M盐酸胍和70%乙醇来进行清洗。介质的合成工艺稳定，不同批次间的重现性较好。将介质用于大鼠肝脏中谷胱甘肽S转移酶的分离纯化，先用离子交换对大鼠肝匀浆液进行粗分离，将获得的粗分离液用所研制的介质做亲和层析，纯化的谷胱甘肽S转移酶纯度达到电泳纯(95%)，酶活力达到12 U/mg，总回收率达到40%以上。 |
| 英文摘要 | In this study, glutathione (GSH) was coupled to the agarose matrices via covalent linkage to prepare glutathione affinity absorbent, which has been widely used to purify glutathione s-transferase (GST) and GST fusion proteins. Various technological conditions were investigated in preparation of glutathione affinity adsorbents with size-variable spacer arms and controllable ligand. To begin with, GSH affinity adsorbents were prepared by coupling GSH to epoxy-activated agarose produced by 1, 4-butanediol diglycidyl ether (BDGE). In the activation step, the reactive conditions including amount of BDGE and NaBH4, reaction time, temperature and the initial concentration of sodium borohydride, were investigated. In the coupling step, the effects of operating conditions (GSH amount, pH value of the buffer, reaction temperature and time) to the ligand density were evaluated by orthogonal experiments. Optimal conditions for the activation of agarose and subsequent coupling of GSH were achieved. Then, GSH affinity adsorbents with size-variable spacer arms and controllable ligand density were prepared by the optimal conditions for the activation and coupling of GSH. Furthermore, to improve the amount of epoxy groups in the activation, a novel method of preparing high-density epoxy-activated agarose for GSH affinity adsorbents is described. Allylglycidyl ether (AGE) was used as activator to introduce allyl groups which could be “activated” by Br2/H2O to form bromohydroxypropyl groups. And then these bromohydroxypropyl groups are converted to epoxy groups at alkaline pH. Also, the optimal conditions of the epoxy reaction are investigated. The new method minimized the hydrolysis loss of epoxy groups in the BDGE method, and therefore improved the density of the glutathione ligand on the affinity media. The density of epoxy group on the beads was increased from 281μmol/g-dry gel (BDGE) to 660μmol/g-dry gel (AGE). Finally, the physical and chemical stability of affinity adsorbents were also evaluated. The result implied that the adsorbents had excellent mechanical stability, resistant to high pressure and ideal chemical stability to withstand 6M guanidine hydrochloride and 70% ethanol during derivatization; the adsorbents had favorable reproducibility indicating purification efficiency from batch to batch by the same synthesis route. By the combination of two-step chromatography, DEAE ion exchange chromatography as a pre-treatment step and the GSH affinity absorbent, the purified GST from rat livers were obtained with the purity of 95%, GST specific activity of 12 U/mg and the total recovery of more than 40%, respectively. |
| 语种 | 中文 |
| 公开日期 | 2013-09-13 |
| 页码 | 93 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1145]  |
| 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 | 张海龙. 谷胱甘肽亲和吸附介质的制备及其对谷胱甘肽硫转移酶的纯化[D]. 过程工程研究所. 中国科学院过程工程研究所. 2007. |
入库方式： OAI收割
来源：过程工程研究所
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