Dataset Information

OEPR Cloning: an Efficient and Seamless Cloning Strategy for Large- and Multi-Fragments.

ABSTRACT: Here, an efficient cloning strategy for large DNA fragments and for simultaneous assembly of multiple DNA fragments assembly is presented. This strategy is named OEPR (based on Overlap Extension PCR and Recombination in vivo). OEPR cloning is a seamless, restriction- and ligation-independent method. The method takes advantage of both homologous recombination enzymes in E. coli and overlap PCR. Using OEPR cloning, a long fragment (1-6?kb) or multiple fragments (2-4 fragments) can be easily constructed and simultaneously assembled into a target vector.

SUBMITTER: Liu CJ

PROVIDER: S-EPMC5353728 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Publications

OEPR Cloning: an Efficient and Seamless Cloning Strategy for Large- and Multi-Fragments.

Liu Chang-Jun CJ Jiang Hui H Wu Lei L Zhu Ling-Yun LY Meng Er E Zhang Dong-Yi DY

Scientific reports 20170316

Here, an efficient cloning strategy for large DNA fragments and for simultaneous assembly of multiple DNA fragments assembly is presented. This strategy is named OEPR (based on Overlap Extension PCR and Recombination in vivo). OEPR cloning is a seamless, restriction- and ligation-independent method. The method takes advantage of both homologous recombination enzymes in E. coli and overlap PCR. Using OEPR cloning, a long fragment (1-6 kb) or multiple fragments (2-4 fragments) can be easily constr ...[more]

PMID: 28300166

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Project description:BackgroundTagging proteins is a standard method facilitating protein detection, purification or targeting. When tagging a certain protein of interest, it is challenging to predict which tag will give optimal results and will not interfere with protein folding, activity or yields. Ideally, multiple tags and positions are tested which however complicates molecular cloning and expression vector generation. In conventional cloning, tags are either added on PCR primers (requiring a distinct primer and PCR product per tag) or provided on the vector (typically leaving a restriction site scar).ResultsHere we report a vector family of 40 plasmids allowing simple, seamless fusions of a single PCR product with various N- and C-terminal tags, signal sequences and promoters. The restriction site free cloning (RSFC) strategy presented in this paper relies on seamless cloning using type IIS restriction endonucleases. After cutting out a stuffer (placeholder) fragment from the vectors, a single PCR product can be directly inserted in frame into all 40 plasmids using blunt end or TA ligations, requiring only verification of the orientation. We have established a RSFC vector family for the commonly used protein expression host Pichia pastoris and demonstrated the system with the secretory expression of horseradish peroxidase (HRP). HRP fusions to four tags (Myc, FLAG, His, Strep) and two fusion proteins (GFP and MBP) showed a 31-fold difference in volumetric activities. C-terminal tagging caused in some cases almost a complete loss of function, whereas N-terminal tags showed moderate differences.ConclusionsThe RSFC vectors provide an unprecedented toolbox for expression optimization in P. pastoris. The results obtained with HRP underline the importance of comparing different tags to maximize activities of fusion proteins. In a similar fashion the RSFC strategy can be applied in other expression hosts to screen for optimal promoters, signal sequences or to facilitate the evaluation of (iso-) enzyme families.

Dataset Information

OEPR Cloning: an Efficient and Seamless Cloning Strategy for Large- and Multi-Fragments.

Publications

OEPR Cloning: an Efficient and Seamless Cloning Strategy for Large- and Multi-Fragments.

Similar Datasets

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