Basic fibroblast growth factor (bFGF) is a multi-potential growth factor whose biological activities depend on its receptor’s intrinsic tyrosine kinase activity and second messengers such as the mitogen activated pro...Basic fibroblast growth factor (bFGF) is a multi-potential growth factor whose biological activities depend on its receptor’s intrinsic tyrosine kinase activity and second messengers such as the mitogen activated protein kinases. Heparin sulfate proteoglycans (HSPGs) have been demonstrated to enhance or inhibit bFGF activity. The response elicited by HSPG is related to the relative concentrations and binding kinetics for bFGF of the various pools of HSPG. The type of cellular response might depend on the specific HSPG and FGF receptor expressed on the cell surface. The specific core protein of HSPG, and tissue specific differences in heparin sulfate modification result in altered bFGF regulation.展开更多
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...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.展开更多
目的:高效表达和制备有活性的人表皮生长因子(hum an ep iderm al growth factor,hEGF)。方法:用DNA重组技术构建EGF基因并插入表达载体pQE-40,与载体上的6×H is标签融合,获得的表达质粒pQE-EGF转化E.coliM15[pREP4],得工程菌M15[p...目的:高效表达和制备有活性的人表皮生长因子(hum an ep iderm al growth factor,hEGF)。方法:用DNA重组技术构建EGF基因并插入表达载体pQE-40,与载体上的6×H is标签融合,获得的表达质粒pQE-EGF转化E.coliM15[pREP4],得工程菌M15[pQE-EGF]。MTT法测定活性,培养工程菌M15[pQE-EGF]并诱导目的蛋白表达,镍离子螯合亲和层析纯化重组EGF。结果:所构建的EGF序列正确,重组EGF在大肠杆菌中以包涵体形式存在,表达量占菌体总蛋白的21.2%,经镍离子亲和层析一步纯化后的重组EGF纯度达90%,得率为94%。复性后的重组EGF具有促Hela细胞生长活性。结论:以E.coliM15[pREP4]/pQE-40系统表达EGF具有技术路线简单、目的蛋白得率高、成本低等优点,是一种制备活性hEGF的有效手段。展开更多
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...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。展开更多
文摘Basic fibroblast growth factor (bFGF) is a multi-potential growth factor whose biological activities depend on its receptor’s intrinsic tyrosine kinase activity and second messengers such as the mitogen activated protein kinases. Heparin sulfate proteoglycans (HSPGs) have been demonstrated to enhance or inhibit bFGF activity. The response elicited by HSPG is related to the relative concentrations and binding kinetics for bFGF of the various pools of HSPG. The type of cellular response might depend on the specific HSPG and FGF receptor expressed on the cell surface. The specific core protein of HSPG, and tissue specific differences in heparin sulfate modification result in altered bFGF regulation.
文摘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.
文摘目的:高效表达和制备有活性的人表皮生长因子(hum an ep iderm al growth factor,hEGF)。方法:用DNA重组技术构建EGF基因并插入表达载体pQE-40,与载体上的6×H is标签融合,获得的表达质粒pQE-EGF转化E.coliM15[pREP4],得工程菌M15[pQE-EGF]。MTT法测定活性,培养工程菌M15[pQE-EGF]并诱导目的蛋白表达,镍离子螯合亲和层析纯化重组EGF。结果:所构建的EGF序列正确,重组EGF在大肠杆菌中以包涵体形式存在,表达量占菌体总蛋白的21.2%,经镍离子亲和层析一步纯化后的重组EGF纯度达90%,得率为94%。复性后的重组EGF具有促Hela细胞生长活性。结论:以E.coliM15[pREP4]/pQE-40系统表达EGF具有技术路线简单、目的蛋白得率高、成本低等优点,是一种制备活性hEGF的有效手段。
文摘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。
