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首页> 外文期刊>International Journal of Biological Macromolecules: Structure, Function and Interactions >L-tryptophan and dipeptide derivatives for supercoiled plasmid DNA purification
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L-tryptophan and dipeptide derivatives for supercoiled plasmid DNA purification

机译：L-色氨酸和二肽衍生物用于超螺旋质粒DNA纯化

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摘要

The present study focus on the preparation of chromatography supports for affinity-based chromatography of supercoiled plasmid purification. Three L-tryptophan based supports are prepared through immobilization on epoxy-activated Sepharose and characterized by HR-MAS NMR. The SPR is employed for a fast screening of L-tryptophan derivatives, as potential ligands for the biorecognition of supercoiled isoform, as well as, to establish the suitable experimental conditions for the chromatography. The results reveal that the overall affinity is high (K-D =10(-9) and 10(-8) M) and the conditions tested show that the use of HEPES 100 mM enables the separation and purification of supercoiled at T=10 degrees C. The STD-NMR is performed to accomplish the epitope mapping of the 5'-mononucleotides bound to L-tryptophan derivatives supports. The data shows that the interactions between the three supports and the 5'-mononucleotides are mainly hydrophobic and pi-pi stacking. The chromatography experiments are performed with L-tryptophan support and plasmids pVAX-LacZ and pPH600. The supercoiled isoform separation is achieved at T=10 degrees C by decreasing the concentration of (NH4)(2)SO4 from 2.7 to 0 M in HEPES for pVAX-LacZ and 2.65 M to 0 M in HEPES for pPH600.
机译：本研究集中于层析支持物的制备，以用于超螺旋质粒纯化的基于亲和力的层析。通过固定在环氧活化的琼脂糖上制备三种基于L-色氨酸的载体，并通过HR-MAS NMR进行表征。 SPR用于快速筛选L-色氨酸衍生物，作为超螺旋亚型生物识别的潜在配体，并为色谱建立合适的实验条件。结果表明，总体亲和力很高（​​KD = 10（-9）和10（-8）M），测试条件表明，使用HEPES 100 mM可以分离和纯化T = 10℃的超螺旋进行STD-NMR以完成与L-色氨酸衍生物载体结合的5'-单核苷酸的表位作图。数据显示，三种支持物与5'-单核苷酸之间的相互作用主要是疏水性和pi-pi堆积。用L-色氨酸支持物和质粒pVAX-LacZ和pPH600进行色谱实验。对于pVAX-LacZ，在HEPES中将（NH4）（2）SO4的浓度从2.7降低到0 M，对于pPH600在HEPES中将2.65 M降低到0 M，可以在T = 10摄氏度下实现超螺旋同工型分离。

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