Development of a novel Gateway-based vector system for efficient, multiparallel protein expression in Escherichia coli.
We describe a cloning and expression system which is based on the Escherichia coli T7 expression system and Gateway recombination technology. We have produced numerous destination vectors with selected fusion tags and an additional set of entry vectors containing the gene of interest and optional labeling tags. This powerful system enables us to transfer a cDNA to several expression vectors in parallel and combine them with various labeling tags. To remove the attached amino terminal tags along with the unwanted attB1 site, we inserted PreScission protease cleavage sites. In contrast to the commercially available destination vectors, our plasmids provide kanamycin resistance, which can be an advantage when expressing toxic proteins in E. coli. Some small-scale protein expression experiments are shown to demonstrate the usefulness of these novel Gateway vectors. In summary, this system has some benefits over the widely used and commercially available Gateway standard system, and it enables many different combinations for expression constructs from a single gene of interest.
Freuler
Felix
F
Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry (CPC), Forum 1, Novartis Campus, CH-4056 Basel, Switzerland. felix.freuler@novartis.com
Stettler
Thomas
T
Meyerhofer
Marco
M
Leder
Lukas
L
Mayr
Lorenz M
LM
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins