Abstract
Vibrio mimicus arylesterase, a 20 kDa protein, is a multifunctional enzyme with thioesterase and chymotrypsin-like activities. Because an affinity His-tag (six consecutive histidine affinity tag) directly to the protein caused the loss of enzyme activity, a hexadecapeptide with His-tag, ADPNSSSVDKLAAALEHHHHHH encoded from vector pET-20b(+) was constructed to extend from the carboxyl terminus of the arylesterase. This Histagged protein retained enzyme functions. Thermal unfolding behavior of both proteins was almost identical, and their T m values were near 54°C as monitored by circular dichroism. Tryptic cleavage of the functional His-tagged enzyme produced two smaller proteins, which still possessed enzyme activity and which suggested that the additional peptide extended on the protein surface. The spacing peptide between His-tag and arylesterase successfully prevented the interference of the His-tag to the enzyme functions. The kinetic studies showed that the esterase and thioesterase activities of the His-tagged enzyme were 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.
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Lee, YL., Chang, RC. & Shaw, JF. Facile purification of a C-terminal extended His-tagged Vibrio mimicus arylesterase and characterization of the purified enzyme. J Amer Oil Chem Soc 74, 1371–1376 (1997). https://doi.org/10.1007/s11746-997-0239-1
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DOI: https://doi.org/10.1007/s11746-997-0239-1