Abstract
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.
Highlights
Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts
The Epstein-Barr virus nuclear antigen 1 (EBNA1) in HEK293E cell line interacts with oriP on pTT vectors which increases plasmid persistence and protein expression levels [11]
Vector design and cloning strategy In order to drive strong constitutive ccdB expression from pCoofy vectors, we used the promoter of the major outer membrane lipoprotein Outer membrane lipoprotein A (OmpA), which is one of the strongest promoters in E. coli [14]
Summary
Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. We perfom indepth protein analysis to ensure that delivered proteins are Eukaryotic hosts were used if suggested by the literature or previous experience, or in case E. coli screening had failed As this happened frequently we decided to implement parallel testing of constructs, solubility tags and expression hosts altogether. The method must be sequence and restriction-site independent to allow for incorporation of any DNA fragment into any of the vectors It must be directional and precise and most important, it must be fast, simple, efficient and cost-effective. In the SLIC strategy presented here, ccdB introduced into the vector series was designed for strong constitutive expression in order to suppress growth in non-resistant cells at 100% efficiency. We developed pET, pFastBac and pTT parallel cloning vectors, named pCoofy1-x and present protein expression data in each of the respective host organisms
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