High level of soluble expression and purification of catalytically active native UDP-galactose 4-epimerase of <i>Aeromonas hydrophila</i> in <i>E. coli</i> — Oak Academic Publishing
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High level of soluble expression and purification of catalytically active native UDP-galactose 4-epimerase of <i>Aeromonas hydrophila</i> in <i>E. coli</i>
The ubiquitous <i>Aeromonas hydrophila</i> is responsible for several pathological conditions in fish and human. Like most gram negative bacteria, its virulence relies on outer membrane lipopolysachharide (LPS). The Leloir pathway enzyme UDP-galactose 4-epimerase (GalE), plays an important role in the LPS biosynthesis, and therefore is a potential drug target. We have earlier carried out extensive biochemical and biophysical studies with histidine-tagged recombinant GalE. However, for effective drug design it is desirable to understand the structure-function relation of a protein in its native form without any additional sequences or tags. In the present study, we report the high level expression, purification and characterization of recombinant GalE (rGalE) of <i>Aeromonas hydrophila </i>in its native form in <i>E coli</i>. The rGalE expressed as a soluble protein was purified to near homogeneity. From 1 L of shake flask culture ~15 mg of purified rGalE was obtained. The purified protein was biologically active with K<sub>m</sub> and K<sub>cat</sub> values of 0.7 mM and 28.8 s<sup>–1</sup>, respectively. The enzyme exhibited a temperature and pH optima of 37<sup>。</sup>C and 7 - 9, respectively. Thus, the present study employed for soluble expression and purification of functionally active rGalE without any tag bypasses the need for cumbersome strategies associated with removal of tag from purified protein.
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