中试规模高效纯化重组人核苷二磷酸激酶A

Efficient Purification of Recombinant Human NDPK-A in Pilot-scale

摘要:

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NDPK 47.6g，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100 g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。

Abstract:

To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) efficiently in pilot scale, cells of rhNDPK-A producing E. coli were homogenized by high pressure under 4℃, 950 Pa. The insoluble debris was removed by microfiltration and the soluble portion was concentrated by ultrafiltration. The resulted crude sample was loaded on DEAE-sepharose Fast Flow. The target fraction was collected and then load on Cibacron Blue 3GA Sepharose CL-4B. Eluted with buffer containing ATP from the AC column, rhNDPK-A was polished with ultrafiltration. The results showed that after homogenized 2 rounds, 1500g cells of E. coli brought crude sample containing 47.6g NDPK-A. Treated with microfiltration and ultrafiltration, 27.3g of NDPK-A were recovered from this bacteria homogenate. After 2-step purification with column chromatography and then polished with ultrafiltration, 17.2 g rhNDPK-A were collected with purity of 96.3%. The recovery of the whole purification process was 36.2%, and the productivity of rhNDPK-A was 1.15 g per 100 g wet cells. Comparing the recovery of each purification step, it was found that the recovery of polish is higher than that of affinity chromatography, which is higher than that of ion exchange chromatography. The limit step was the process of sample pretreatment among the 4 purification steps. Combine with the fermentation results reported before, it was deduced that the productivity of rhNDPK-A was 510 mg/L. In conclusion, an easily controlled purification condition with high yield provides material for the translation researches of NDPK; In addition, it was suggested the crucial step determine the recovery of non-secretive recombinant proteins might be the process of sample pretreatment, not be the process of column chromatography.