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Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain ofPichia pastoris transformed with a plasmid, bearing multiple ENP gene copies.The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter. Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp. These were then integrated at the HIS locus of P. pastoris GS115 (his4).Clones were chosen for their ability to produce rENP upon methanol induction in shaker flasks, and then the 1x, 2x, and 3x-enp strains were analyzed by Southern blot for the presence of the ENP gene(s). Isolate 3x5q, containing a 3x-enp cassette, was the best producer of rENP. Under optimal conditions this strain grown in a fed-batch mode produced yields of >500mg rENP/Lwith an average of 5.46mg rENP/g DCW. Purification of rENP from the clarified broth resulted in a yield of 35% and a purity of >86%. Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized. The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene.2002 Elsevier Science (USA). All rights reserved. |
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Clones containing a single enp insert were used to construct cassettes bearing 2 and 3 tandem copies of enp. These were then integrated at the HIS locus of P. pastoris GS115 (his4). Clones were chosen for their ability to produce rENP upon methanol induction in shaker flasks, and then the 1x, 2x, and 3x-enp strains were analyzed by Southern blot for the presence of the ENP gene(s). 含有单个ENP插入物的克隆被用来构建含有2或3个连续ENP的拷贝框。然后把他们整合到毕赤酵母GS115组氨酸位点。在摇瓶中中，根据克隆在甲醇诱导下生产rENP的能力进行选择。然后用1X、2X、3XENP菌株通过Southern杂交来检测ENP基因是否存在。 Isolate 3x5q, containing a 3x-enp cassette, was the best producer of rENP. Under optimal conditions this strain grown in a fed-batch mode produced yields of >500mg rENP/Lwith an average of 5.46mg rENP/g DCW. Purification of rENP from the clarified broth resulted in a yield of 35% and a purity of >86%. 包含有3XENP框的独立3x5q（这里实在是不知道怎么翻译了，应该是个菌种品系吧），是生产rENP最好的选择。在可视条件下，这个菌株在连续补加培养模式下生长生产产量和每克5.46毫克 rENP平均干菌量想比会产生大于500毫克每升的rENP。 |
3楼2011-04-08 15:48:27
wd519wd706(金币+20, 翻译EPI+1): 感谢你 2011-04-09 02:17:40
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Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification 毕赤酵母中重组内毒素中和蛋白的生产及其纯化方法。 Production of recombinant Limulus endotoxin neutralizing protein (rENP) was attained with the GS115 methylotrophic strain of Pichia pastoris transformed with a plasmid, bearing multiple ENP gene copies. The synthetic gene for Limulus ENP was cloned into the integrative plasmid pAO815 under the control of a methanol-inducible promoter. 通过使用带有多种ENP基因拷贝的质粒转染GS115甲醇毕赤酵母菌株，从而获得鲎重组内毒素中和蛋白产物。鲎ENP的合成基因在甲醇诱导启动子调控下被克隆到整合质粒pAO815上。 |
2楼2011-04-08 15:20:09
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Glycosylated rENP, the main contaminant, was removed with a concanavalin-A column and characterized. The pure rENP neutralized lipopolysaccharide and had the mass, amino-acid composition and N-terminal sequence expected from the cloned gene.2002 Elsevier Science (USA). All rights reserved. 主要的重组形式：糖基化rENP，可以被伴刀豆球蛋白亲和色谱柱转移并标记。纯化的rENP能够中和脂多糖并且含有大量氨基酸成分和从克隆基因中所需要的N端序列。 |
4楼2011-04-08 15:54:59
