FACILE PURIFICATION OF A C-TERMINAL EXTENDED HIS-TAGGED VIBRIO-MIMICUS ARYLESTERASE AND CHARACTERIZATION OF THE PURIFIED ENZYME

Vibrio mimicus arylesterase, a 20 kDa protein, is a multifunctional en zyme with thioesterase and chymotrypsin-like activities. Because an af finity His-tag (six consecutive histidine affinity tag) directly to th e protein caused the loss of enzyme activity, a hexadecapeptide with H is-tap, ADPNSSSVDKLAAALEHHHHHH encoded from vector pET-20b(+) was cons tructed to extend from the carboxyl terminus of the arylesterase. This His-tagged protein retained enzyme functions. Thermal unfolding behav ior of both proteins was almost identical, and their T-m values were n ear 54 degrees C as monitored by circular dichroism. Tryptic cleavage of the functional His-tagged enzyme produced two smaller proteins, whi ch still possessed enzyme activity and which suggested that the additi onal peptide extended on the protein surface. The spacing peptide betw een His-tag and arylesterase successfully prevented the interference o f the His-rag to the enzyme functions. The kinetic studies showed that the esterase and thioesterase activities of the His-tagged enzyme wer e similar to those of the wild type. On the other hand, the catalytic efficiency of chymotrypsin-like activity of the His-tagged protein was two times higher than that of the wild type.