TAGZyme pQE Vector Set

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Protein Expression and Purification: For expression of 6xHis-tagged proteins in Escherichia coli, cDNAs were cloned into expression vectors pQE-30 and TAGZyme pQE-1 and -2 using standard PCR-based techniques,21–23 sequenced, trans- formed into E.coli M15/pREP4, and plated on appro- priate selective LB agar. Constructs created for this work are described in Table 1. Single clones showing high quanitative expression rates were selected by the colony blot procedure.22 The clones showing the highest expression rates were chosen for glycerol stock preparation and used to inoculate growth cul- tures in shake flasks or small fermenters (Biostat B5, B. Braun Biotech, Melsungen, Germany). Proteins were purified from E.coli pellets by Ni-NTA IMAC under native conditions as previously described.22 EDTA (10 mM) was added to protein eluates to chelate traces of heavy metal ions, and protein sam- ples were dialyzed versus TAGZyme buffer (20 mM sodium phosphate; 150 mM NaCl, pH 7.0).


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