谷胱甘肽亲和层析介质的制备条件及其层析特性研究

The Preparation of GSH Affinity Chromatographic Adsorbent and the Research of its Chromatographic Characteristics

以琼脂糖凝胶4FF（Sepharose 4 Fast Flow）为基质,1,4-丁二醇二缩水甘油醚（BDGE）为活化剂,通过活化、偶联谷胱甘肽（GSH）,得到GSH亲和层析介质.实验结果表明：活化及偶联的优化条件为10,m L基质加入0.42,g Na OH、15,m L二甲基亚砜（DMSO）、15,m L BDGE,反应温度为40,℃,反应时间为6,h;偶联溶液p H 6.5,反应温度为37,℃,反应时间为24,h.在此条件下,环氧基修饰密度可达60~65,μmol/m L;所制备的介质载量为（14.19±0.98）mg/m L.以此介质对谷胱甘肽S–转移酶（GST）、融合蛋白GST-Bcl-2、GST-Ald、GST-PTPN12进行纯化,结果表明,该介质纯化效果较好、载量高、稳定性能较好,可重复使用.

The glutathione （GSH）affinity adsoebent was prepared with Sepharose 4 Fast Flow and 1,4-bntanediol digly- cidyl ether （BDGE）, and then coupled with GSH based on the activated agarose particles. The results showed that the activat- ing and coupling optimization conditions were adding into 10 mL of Sepharose 4 Fast Flow 0.42 g of NaOH, 15 mL of di- methyl sulfoxide （DMSO） and 15 mL of BDGE. The incubation was at 40 ℃ for 6 h. The activated agarosed particles were coupled with GSH at 37 ℃for 24 h. The pH of the coupling reaction solution was 6.5. Under these conditions, the density of the epoxy group of the agarosed particles was 60-65 μmol/mL and the loading capacity was optimized to be （14.19 ± 0.98） mg/mL. The adsorbents were used to purify the gultathione S transferase （GST）, the recombinant GST-Bcl-2, GST-Ald and GST-PTPN12 proteins. The result shows that the GSH affinity adsorbent has good purification effect and a high loading ca- pacity, and its good stability in performance can make the adsorbent be used repeatedly.