Max-Planck Institute of Biochemistry, Am Klopferspitz 18, Martinsried 82152, Germany.
BMC Biotechnol. 2013 Feb 14;13:12. doi: 10.1186/1472-6750-13-12.
Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector.
Here we describe a method to tailor selected expression vectors for parallel Sequence and Ligation Independent Cloning. SLIC cloning enables precise and sequence independent engineering and is based on joining vector and insert with 15-25 bp homologies on both DNA ends by homologous recombination. We modified expression vectors based on pET, pFastBac and pTT backbones for parallel PCR-based cloning and screening in E.coli, insect cells and HEK293E cells, respectively. We introduced the toxic ccdB gene under control of a strong constitutive promoter for counterselection of insert less vector. In contrast to DpnI treatment commonly used to reduce vector background, ccdB used in our vector series is 100% efficient in killing parental vector carrying cells and reduces vector background to zero. In addition, the 3' end of ccdB functions as a primer binding site common to all vectors. The second shared primer binding site is provided by a HRV 3C protease cleavage site located downstream of purification and solubility enhancing tags for tag removal. We have so far generated more than 30 different parallel expression vectors, and successfully cloned and expressed more than 250 genes with this vector series. There is no size restriction for gene insertion, clone efficiency is > 95% with clone numbers up to 200. The procedure is simple, fast, efficient and cost-effective. All expression vectors showed efficient expression of eGFP and different target proteins requested to be produced and purified at our Core Facility services.
This new expression vector series allows efficient and cost-effective parallel cloning and thus screening of different protein constructs, tags and expression hosts.
正确折叠蛋白质的表达和纯化通常需要筛选不同的参数，如蛋白质变体、提高可溶性的标签或表达宿主。虽然有涵盖所有变化的平行载体系列，但这并非没有折衷。我们已经建立了一种快速、高效且绝对无背景的克隆方法，可应用于任何选定的载体。
在这里，我们描述了一种针对平行序列和连接独立克隆（SLIC）定制选择表达载体的方法。SLIC 克隆能够实现精确的、无需序列信息的工程操作，其基础是通过同源重组将载体和插入片段的 DNA 两端具有 15-25 个碱基对的同源性进行连接。我们基于 pET、pFastBac 和 pTT 骨架修改了表达载体，以便分别在大肠杆菌、昆虫细胞和 HEK293E 细胞中进行基于 PCR 的平行克隆和筛选。我们在强组成型启动子的控制下引入了有毒的 ccdB 基因，用于对抗不含插入片段的载体的选择。与通常用于减少载体背景的 DpnI 处理不同，我们载体系列中的 ccdB 在杀死携带亲本载体的细胞方面效率达到 100%，并将载体背景降低到零。此外，ccdB 的 3' 端作为所有载体的引物结合位点。第二个共享的引物结合位点由位于纯化和提高可溶性标签下游的 HRV 3C 蛋白酶切割位点提供，用于去除标签。迄今为止，我们已经生成了 30 多种不同的平行表达载体，并成功地用这个载体系列克隆和表达了 250 多个基因。基因插入没有大小限制，克隆效率>95%，克隆数量可达 200 个。该程序简单、快速、高效且具有成本效益。所有表达载体都能有效地表达 eGFP 和我们核心设施服务请求生产和纯化的不同目标蛋白。
这个新的表达载体系列允许高效且具有成本效益的平行克隆，从而筛选不同的蛋白质构建体、标签和表达宿主。
