中试规模纯化重组人核苷二磷酸激酶A(rhNDPK-A)。菌体高压匀浆,然后微滤去除菌体碎片,超滤浓缩,所得样品上样DEAE-sepharose Fast Flow,收集目的峰,上样Cibacron Blue 3GA Sepharose CL-4B,含ATP的缓冲液洗脱目的蛋白,超滤精制。结果表明,1500g菌体经过2次匀浆后,所得匀浆液中含NDPK47.6g,经过微滤超滤处理后,可回收目的蛋白27.3g。再经过两步柱层析及超滤精制后,最终可得纯度为96.3%的目的蛋白17.2g,总回收率为36.2%,每100g湿菌体的蛋白产率为1.15g。比较每个步骤的回收率,发现精制>亲和层析>离子交换层析>样品前处理过程。与前期报道发酵工艺联用,rhNDPK-A的纯化产量达到510mg/L。工艺简便、得率高的rhNDPK-A纯化工艺的建立为NDPK的应用开发提供了物质基础;另外,本文结果也提示,对于非分泌型重组蛋白来说,影响目的蛋白回收的最主要因素可能不是柱层析,而是样品前处理过程。
Human Nm23 family consists of eight gene products that have been implicated in cellular differentiation,development and apoptosis,as well as oncogenesis and tumor metastasis.The protein products of Nm23 are nucleoside diphosphate kinases(NDPKs),the key metabolic enzymes that catalyze the synthesis of nucleoside triphosphates(NTP) by transfer of the terminal phosphate between NDP and NTP.Recent investigations are focused on the extraordinary pleiotropy and its mechanism of Nm23/NDPK.In this article,we review the recent progress in studies of the mechanism of Nm23/NDPK as metastasis suppressors,and other bioactivities of NDPK out of phosphate transfer enzyme,as well as the character of NDPK’s quaternary structure and the relation between its structure and function.In addition to these,the potential applications of NDPK as an enhancer of antiviral drugs or Nm23 as a drug target were also described.Findings herein summarized provide new and intriguing suggestions for a more extensive understanding of the biological functions of the star molecule,Nm23/NDPK.
