Preparation, optimization and application of affinity absorbent with a polysaccharide YCP as the ligand.
Preparation, optimization and application of affinity absorbent with a polysaccharide YCP as the ligand.
复制标题
DOI：
10.1016/j.carbpol.2014.01.005
复制
发表时间：
2014-04
影响因子：
11.2
通讯作者：
Ran Ding;Yan Zhou;Xian Zhang;Rui Zhu;W. Yao;Xiangdong Gao
中科院分区：
文献类型：
--
作者：
Ran Ding;Yan Zhou;Xian Zhang;Rui Zhu;W. Yao;Xiangdong Gao
YCP是从海洋丝状真菌Phoma herbarumYS4108的菌丝体中提取的一种α-葡聚糖，通过Toll样受体2和TLR4信号通路增强宿主免疫，具有很强的抗肿瘤活性。本研究以1-氰基-4-二甲氨基吡啶四氟硼酸盐(CDAP)为活化剂，将YCP偶联到EAH Sepharose4B琼脂糖珠上，制备了YCP-Sepharose亲和吸附剂。采用L9(3)4正交试验对偶联工艺进行了优化，得到的最佳工艺参数为：CDAP与YCP的摩尔比为1：2，CDAP活化时间为5min，凝胶体积为0.1nL，凝胶孵育时间为72h。扫描电子显微镜分析表明，成功地制备了YCP固定化琼脂糖球，而这些小球基本上保持了游离YCP的生物学性质，因为它们可以与TLR2和TLR4在可比水平上特异地相互作用。总而言之，我们的发现为研究碳水化合物-蛋白质相互作用提供了一种固定碳水化合物分子的替代方法。
YCP, an α-glucan from the mycelium of marine filamentous fungusPhoma herbarumYS4108, has great antitumor potential via enhancement of host immune through Toll-like receptor (TLR) 2 and TLR4 signaling. In the current study, YCP was coupled to EAH Sepharose 4B agarose beads to prepare the YCP-Sepharose affinity absorbent using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as the activating agent. An orthogonal experiment L9(3)4was applied to optimize the coupling procedure, giving the optimal parameters as follows: molar ratio of CDAP to YCP of 1:2, CDAP-activation time of 5 min, gel volume of 0.1 mL, and gel-incubation time of 72 h, respectively. Scanning electron microscopy analysis indicated successfully preparation of YCP immobilized sepharose beads, while these beads essentially maintained biological properties of free YCP since they can interact with TLR2 and TLR4 specifically at comparable level. Collectively, our findings provide an alternative approach to immobilize carbohydrate-based molecules for studying the carbohydrate–protein interaction.