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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). 含有單個ENP插入物的克隆被用來構(gòu)建含有2或3個連續(xù)ENP的拷貝框。然后把他們整合到畢赤酵母GS115組氨酸位點。在搖瓶中中，根據(jù)克隆在甲醇誘導下生產(chǎn)rENP的能力進行選擇。然后用1X、2X、3XENP菌株通過Southern雜交來檢測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框的獨立3x5q（這里實在是不知道怎么翻譯了，應該是個菌種品系吧），是生產(chǎn)rENP最好的選擇。在可視條件下，這個菌株在連續(xù)補加培養(yǎng)模式下生長生產(chǎn)產(chǎn)量和每克5.46毫克 rENP平均干菌量想比會產(chǎn)生大于500毫克每升的rENP。 |
3樓2011-04-08 15:48:27
wd519wd706(金幣+20, 翻譯EPI+1): 感謝你 2011-04-09 02:17:40
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Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification 畢赤酵母中重組內(nèi)毒素中和蛋白的生產(chǎn)及其純化方法。 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. 通過使用帶有多種ENP基因拷貝的質(zhì)粒轉(zhuǎn)染GS115甲醇畢赤酵母菌株，從而獲得鱟重組內(nèi)毒素中和蛋白產(chǎn)物。鱟ENP的合成基因在甲醇誘導啟動子調(diào)控下被克隆到整合質(zhì)粒pAO815上。 |
2樓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. 主要的重組形式：糖基化rENP，可以被伴刀豆球蛋白親和色譜柱轉(zhuǎn)移并標記。純化的rENP能夠中和脂多糖并且含有大量氨基酸成分和從克隆基因中所需要的N端序列。 |
4樓2011-04-08 15:54:59
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