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A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

机译:适用于高通量表达筛选应用的通用的不依赖连接的克隆方法

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摘要

This article describes the construction of a set of versatile expression vectors based on the In-Fusion™ cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion™ has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His6-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.
机译:本文介绍了基于In-Fusion™克隆酶的一组通用表达载体的构建及其在高通量克隆和表达筛选中的用途。对常用载体的修饰使其与In-Fusion™兼容,从而产生了非连接依赖性的克隆系统,该系统具有(1)不依赖插入序列的功能(2)能够克隆宽大(20倍)的大型PCR片段(3)插入浓度范围和(4)适用于在多个宿主中表达。该系统无需将不希望的载体或限制性位点衍生的氨基酸添加到表达的蛋白质中,即可对(His6-)标记的构建体进行精确工程设计。使用支持多种宿主的载体可以在大肠杆菌和真核宿主(HEK293T细胞和昆虫细胞宿主,例如Sf9细胞)中进行快速筛选。这些高通量的筛选活动已促使开发和验证用于转染哺乳动物细胞和Ni-NTA蛋白纯化的自动化方案。

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