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pET-44 Ek/LIC Vector Kit
Production of soluble, active proteins in E. coli
pET-44 Ek/LIC Vector Kit
pET Expression Systems and pET Expression Systems plus Competent Cells provide core reagents needed for target gene cloning and expression.The pET-43.1 vectors incorporate the Nus•Tag fusion partner and are also compatible with the trxB/gor mutant Origami, Origami 2, Origami B, Rosetta-gami, Rosetta-gami 2, and Rosetta-gami B strains, which facilitatedisulfide bond formation in the cytoplasm (Davis 1998). Disulfide-bonded proteins maybe obtained by employing the combination of a pET-43.1 or pET-44 vector and these trxB/gor host strains. Unfused proteins can be produced by using the Nde I cloning site.
Directional PCR cloning into the most powerful E. coli expression vectors
The pET-44 vectors incorporate the Nus•Tag™ technology, which allows target proteins to be expressed as fusions to the highly soluble NusA protein. According to a modeling study, the NusA protein has the highest potential for solubility among more than 4000 E. coli proteins, and therefore increases the proportion of soluble protein when expressed in E. coli as a fusion partner (1, 2). Ek/LIC represents a highly flexible cloning strategy in which a single insert preparation can be simultaneously cloned into a wide variety of compatible Ligation Independent Cloning (LIC) vectors to allow high-throughput optimization of target protein expression (3). The Ek/LIC Nus•Tag vector, pET-43.1 Ek/LIC, carries an N-terminal Nus•Tag/His•Tag®/ S•Tag™ configuration, followed by protease cleavage sites, multiple cloning site region and optional C-terminal HSV•Tag® and His•Tag sequences. The new pET-44 Ek/LIC Vector encodes an additional His•Tag peptide at the N-terminus but is otherwise identical to pET-43.1 Ek/LIC. We have shown that the additional N-terminal His•Tag significantly enhances purification yields when utilizing Novagen's His•Mag™ Agarose Beads for high-throughput purification (4).
The various pET Ek/LIC Vectors are shown below. Configurations of the pCDF-2 and pRSF-2 Ek/LIC Vectors, which are designed for coexpression, are also shown. For additional information about the LIC method and Ek/LIC primer design, refer to the previous section, The LIC Method. Ek/LIC vectors for insect and mammalian expression are also available.
The pET System allows for expression of target genes under control of the T7 promoter and is covered by US Patent 5,693,489. This patent is licensed by EMD Millipore Corporation from Brookhaven National Labs. Commercial entities need to obtain a license from Brookhaven National Labs prior to use of this Product. Ccommercial use of this Product also requires a commercial license from EMD Millipore Corporation. Commercial use shall include but not be limited to (1) use of the Product or its components in manufacturing; (2) use of the Product or its components to provide a service, information, or data to others in exchange for consideration; (3) use of the Product or its components (or any derivatives thereof) for therapeutic, diagnostic or prophylactic purposes (including as part of a device, chip, assay or other product); or (4) resale of the Product or its components, whether or not such Product or its components are resold for use in research. Nothing contained herein shall be deemed to represent or warrant that additional third party rights are not required for use of this Product.
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产品信息
Components
• 1 µg pET-44 Ek/LIC Vector
• 8 µl Ek/LIC b-Gal Control Insert
• 25 U T4 DNA Polymerase, LIC-qualified
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