Directional PCR cloning into the most powerful E. coli
expression vectorsThe 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). Novagen's original 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).