A new method to customize protein expression vectors for fast, efficient and background free parallel cloning

BMC Biotechnology

Methodology Article

Hüseyin Besir¹  Claudia Strasser²  Sabine Suppmann²  Judith Scholz² 

[1] EMBL Heidelberg, Meyerhofstrasse 1, 69117, Heidelberg, Germany;Max-Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany;

关键词: Parallel cloning;    Restriction enzyme free cloning;    Expression screening;    Bacterial expression;    Insect cell expression;    Mammalian expression;    Protein production;   

DOI  :  10.1186/1472-6750-13-12

 received in 2012-08-21, accepted in 2012-12-21,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundExpression 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.ResultsHere 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.ConclusionThis new expression vector series allows efficient and cost-effective parallel cloning and thus screening of different protein constructs, tags and expression hosts.

【 授权许可】

Unknown   
© Scholz et al; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.