公开(公告)号：US5523211A

公开(公告)日：1996-06-04

申请号：US277076

申请日：1994-07-19

申请人： Marianne Pusztai-Carey ,  Paul R. Carey ,  Timothy Lessard ,  Makoto Yaguchi

发明人： Marianne Pusztai-Carey ,  Paul R. Carey ,  Timothy Lessard ,  Makoto Yaguchi

IPC分类号： A01N63/02 ,  C07K14/325 ,  G01N30/02 ,  G01N30/34 ,  G01N33/569 ,  C12Q1/37 ,  C12N1/20 ,  C12Q1/00 ,  C12Q1/24

CPC分类号： G01N33/56911 ,  A01N63/02 ,  C07K14/325 ,  G01N30/02 ,  G01N30/34

摘要： A process for identifying proteinaceous protoxins expressed by Bacillus thuringiensis genes is disclosed. According to the process, daughter toxins are first generated by subjecting a protoxin-containing material, such as parasporal crystals of Bacillus thuringiensis, to limited proteolysis with a proteolytic enzyme in an aqueous suspension having a pH above 9.5. The daughter toxins are then separated by high performance anion-exchange liquid chromatography at a constant pH in excess of 10 in an increasing gradient of a salt, preferably sodium chloride. The gradient conditions, which are specific for the column used, are achieved by employing a series of buffers having increasing concentration of the salt and introduced at a predetermined time and rate. The procedure provides a chromatogram showing clearly identifiable peaks of toxins and permits therefore the qualitative and quantitative characterization of the original mixture and isolation of the individual toxins. By this it provides a way of screening and testing new Bacillus thuringiensis isolates, both single--and multigene, and monitoring the level of expression of known genes from a known strain. The digestion and isolation conditions permit the production of the toxins in a biologically fully active state.

摘要翻译： 公开了一种鉴定苏云金芽孢杆菌基因表达的蛋白质原毒素的方法。 根据该方法，首先通过使含有原毒素的材料（例如苏云金芽孢杆菌的寄生虫晶体）在pH高于9.5的水性悬浮液中用蛋白水解酶限制蛋白水解产生子代毒素。 然后通过高效阴离子交换液相色谱法，在不断增加的盐，优选氯化钠梯度下，以恒定的pH超过10分离子体毒素。 对所使用的柱特异的梯度条件通过使用一系列具有增加的盐浓度并以预定时间和速率引入的缓冲液来实现。 该程序提供了一个色谱图，显示了清晰可辨的毒素峰值，因此允许原始混合物的定性和定量表征以及各种毒素的分离。 因此，它提供了一种筛选和测试新型苏云金芽孢杆菌分离株，单基因和多基因的方法，并监测已知菌株已知基因的表达水平。 消化和分离条件允许在生物学上完全活跃的状态下生产毒素。

公开(公告)号：US5356788A

公开(公告)日：1994-10-18

申请号：US102491

申请日：1993-08-05

申请人： Paul R. Carey ,  Timothy Lessard ,  Makoto Yaguchi ,  Marianne Pusztai

发明人： Paul R. Carey ,  Timothy Lessard ,  Makoto Yaguchi ,  Marianne Pusztai

IPC分类号： A01N63/02 ,  C07K1/22 ,  C07K14/325 ,  C12Q1/04 ,  C12Q1/37 ,  C12Q1/68 ,  G01N30/00 ,  G01N30/02 ,  G01N30/34 ,  G01N33/569 ,  C12N1/20 ,  G01N33/567

CPC分类号： C07K14/325 ,  A01N63/02 ,  C12Q1/04 ,  G01N2333/325 ,  G01N30/02 ,  G01N30/34 ,  Y10S435/803

摘要： A process for identifying proteinaceous protoxins expressed by Bacillus thuringiensis genes is disclosed. According to the process, daughter toxins are first generated by subjecting a protoxin-containing material, such as parasporal crystals of Bacillus thuringiensis, to limited proteolysis with a proteolytic enzyme in an aqueous suspension having a pH above 9.5. The daughter toxins are then separated by high performance anion-exchange liquid chromatography at a constant pH in excess of 10 in an increasing gradient of a salt, preferably sodium chloride. The gradient conditions, which are specific for the column used, are achieved by employing a series of buffers having increasing concentration of the salt and introduced at a predetermined time and rate. The procedure provides a chromatogram showing clearly identifiable peaks of toxins and permits therefore the qualitative and quantitative characterization of the original mixture and isolation of the individual toxins. By this it provides means of screening and testing new Bacillus thuringiensis isolates, both single - and multigene, and monitoring the level of expression of known genes from a known strain. The digestion and isolation conditions permit the production of the toxins in a biologically fully active state.

摘要翻译： 公开了一种鉴定苏云金芽孢杆菌基因表达的蛋白质原毒素的方法。 根据该方法，首先通过使含有原毒素的材料（例如苏云金芽孢杆菌的寄生虫晶体）在pH高于9.5的水性悬浮液中用蛋白水解酶限制蛋白水解产生子代毒素。 然后通过高效阴离子交换液相色谱法，在不断增加的盐，优选氯化钠梯度下，以恒定的pH超过10分离子体毒素。 对所使用的柱特异的梯度条件通过使用一系列具有增加的盐浓度并以预定时间和速率引入的缓冲液来实现。 该程序提供了一个色谱图，显示了清晰可辨的毒素峰值，因此允许原始混合物的定性和定量表征以及各种毒素的分离。 因此，它提供了筛选和测试新型苏云金芽孢杆菌分离株单 - 和多基因的方法，并监测已知菌株已知基因的表达水平。 消化和分离条件允许在生物学上完全活跃的状态下生产毒素。