Recombinant pp60c-src from baculovirus-infected insect cells: purification and characterization.
Recombinant pp60c-src from baculovirus-infected insect cells: purification and characterization.
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来自杆状病毒感染的昆虫细胞的重组 pp60c-src：纯化和表征。
DOI：
10.1080/10826069308544572
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Ke,S
中科院分区：
文献类型：
--
作者：
Budde,RJ;Ramdas,L;Ke,S
建立了一种从杆状病毒-昆虫细胞表达系统中纯化重组蛋白酪氨酸激酶pp60c-src的方法。方法包括亲和色谱法和高效液相色谱法。利用底物poly E4Y，分离得到的蛋白质的活性为3.9 μmol/min/mg。这个特定的活动比任何已发布的协议都要高很多倍。酶在- 70°C的缓冲10%甘油中保存数月稳定。将该纯化技术与广泛应用于该酶的免疫亲和技术进行了比较。酶动力学的特征与底物特异性、温度、离子强度、pH和Mg+2对Mn+2离子的影响有关。与在人细胞中表达的酶相似，重组酶对聚E4Y的Vmax和底物特异性高于5V-Agt-II。活化能为14.2 kcal/mol。增加离子强度的抑制作用主要是由于聚E4Y底物Km的增加，因此与底物有关。Km（ATP）与pH有关，而Km（聚E4Y）与pH无关。
A simple and effective method has been developed to purify the recombinant protein tyrosine kinase pp60c-srcfrom a baculovirus-insect cell expression system. The procedure includes affinity chromatography and HPLC. Milligram quantities of protein have been isolated with an activity of 3.9 μmol/min/mg protein using the substrate poly E4Y. This specific activity is many times higher than any published protocol. The enzyme is stable for months when stored in buffered 10% glycerol at −70°C. This purification technique is compared to the immuno-affinity technique which is widely used for this enzyme. Enzyme kinetics were characterized with respect to substrate specificity, the effect of temperature, ionic strength, pH, and Mg+2versus Mn+2ions. Similar to the enzyme expressed in human cells, the recombinant enzyme demonstrated a higher Vmax and substrate specificity for poly E4Y over 5V-Agt-II. An activation energy of 14.2 kcal/mol was determined. Inhibition by increasing ionic strength is mostly due to an increase in Km for the poly E4Y substrate and hence was substrate dependent. The Km(ATP) was pH dependent while the Km(poly E4Y) was pH independent.