LEXSinduce3 Expression Kit (contains the integrative vector pLEXSY_I-blecherry3)

The LEXSinduce3 kit contains one of three expression vectors of choice which differ in the antibiotic resistance marker and the option to monitor expression online by a fluorescence reporter. The vector pLEXSY_I-neo3 (encoding aminoglucoside phosphotransferase) allows selection with the antibiotic LEXSY Neo whereas pLEXSY_I-ble3 (encoding bleomycin resistance gene) permits selection with the antibiotic LEXSY Bleo. The vector pLEXSY_I-blecherry3 facilitates selection with the antibiotic LEXSY Bleo and, in addition, offers the possibility to monitor induction during cultivation by coexpressed fluorescence. The control vector provided in the kit contains the EGFP gene linked to the respective selection marker.

The pLEXSY_I-3 vectors enable inducible expression of target proteins either with or without secretory signal peptide provided on the vectors. For customer convenience the same vector can be used for cloning of ORFs either for cytosolic or for secretory expression. The LmSAP secretory signal peptide encoded on these vectors was derived from the gene for secreted acid phosphatase (lmsap1) of Leishmania mexicana. In-frame fusion of the ORF of a target protein to this signal peptide allows secretory expression in LEXSY hosts, whereas cloning into any of the restriction sites at the 5’ end of the signal peptide-encoding sequence will result in cytosolic expression. An illustration of the inducible LEXSY can be downloaded from the Jena Bioscience website at http://www.jenabioscience.com/images/b3e879b381/Illustration_inducible_LEXSY.pdf

One main advantage of LEXSY is the mammalian-type posttranslational modification of target proteins, such as glycosylation, phosphorylation or prenylation. Recombinant human erythropoietin (EPO) purified at Jena Bioscience from LEXSY was biologically active, natively processed at the N-terminus, and N-glycosylated. The N-glycosylation profile was exceptionally homogeneous, with a biantennary oligosaccharide and the Man3GlcNAc2 core structure accounting for >90% of the glycans present. L. tarentolae is thus the first described biotechnologically useful unicellular eukaryotic host producing biantennary, fully galactosylated, core-α-1,6-fucosylated N-glycans. This N-glycosylation profile was coincident with the profile of recombinant human Interferon-γ expressed in LEXSY and of LEXSY host Gp63 glycoprotein. An illustration of LEXSY glycosylation can be downloaded from the Jena Bioscience website at http://www.jenabioscience.com/images/b3e879b381/Illustration_LEXSY_Glycosylation.pdf.