Chaudhary Rajesh, Ok Lee Jae, Muralidhar K
Hormone Research Laboratory, Department of Zoology, University of Delhi, Delhi, India.
Prep Biochem Biotechnol. 2005;35(4):313-29. doi: 10.1080/10826060500218164.
Loss of buffalo Growth Hormone (buGH) in the various side fractions of standard buGH purification protocol has been determined quantitatively by direct binding ELISA and qualitatively by SDS-PAGE and Western blot analysis. Accounting result indicated that there was a considerable loss of buGH in the side fractions. An alternative protocol to prevent loss and to obtain a high yield of buGH has been developed by introducing anion exchange chromatography, QAE-Sephadex. This has resulted in a simple, reproducible three-step protocol. In this protocol, an extract obtained at 250 mM (NH4)2 SO4, pH 5.5, was loaded onto the QAE-Sephadex column in 0.1 M NH4 HCO3. At this salt concentration, the bulk of the buGH came as QAE unbound fraction. Some amount of buGH, together with contaminating proteins, was bound to QAE-Sephadex and these could be eluted with 1 M KCl. The immunopotency of the enriched buGH preparation "QUB" (QAE unbound fraction) in a direct binding ELISA was similar to that of the semi-pure buGH (ECS/APECS) preparation obtained using the standard protocol, but the yield was 4 times higher. The SDS-PAGE data showed that the banding pattern of standard semi-pure buGH and QUB were quite similar and QUB can be loaded onto the Sephacryl S-200 gel filtration chromatography to yield a highly purified buGH. SDS-PAGE and Western blot analyses showed the major band of buGH in QUB at the same position as in the case of standard buGH. It has also been demonstrated here that it is possible to separate buffalo prolactin (buPRL) and buGH on QAE-Sephadex.
通过直接结合酶联免疫吸附测定法（ELISA）对标准水牛生长激素（buGH）纯化方案各副产物中buGH的损失进行了定量测定，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹分析进行了定性测定。核算结果表明，副产物中存在相当数量的buGH损失。通过引入阴离子交换色谱法（QAE-葡聚糖），开发了一种防止损失并获得高产率buGH的替代方案。这产生了一个简单、可重复的三步方案。在该方案中，将在250 mM硫酸铵（(NH4)2 SO4）、pH 5.5条件下获得的提取物上样到含有0.1 M碳酸氢铵（NH4 HCO3）的QAE-葡聚糖柱上。在此盐浓度下，大部分buGH以QAE未结合级分形式出现。一些buGH与污染蛋白一起结合到QAE-葡聚糖上，这些可以用1 M氯化钾洗脱。富集的buGH制剂“QUB”（QAE未结合级分）在直接结合ELISA中的免疫效力与使用标准方案获得的半纯buGH（ECS/APECS）制剂相似，但产率高出4倍。SDS-PAGE数据表明，标准半纯buGH和QUB的条带模式非常相似，并且QUB可以上样到Sephacryl S-200凝胶过滤色谱柱上以产生高度纯化的buGH。SDS-PAGE和蛋白质免疫印迹分析表明，QUB中buGH的主要条带与标准buGH的位置相同。本文还证明了在QAE-葡聚糖上可以分离水牛催乳素（buPRL）和buGH。
