An improved method for expression of poly-beta-hydroxyalkanoate (
PHA) synthase from Alcaligenes eutrophus has been developed using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) in BTI-TN-5B1-4 Trichoplusia ni cells which results in high level production of active
PHA synthase. Confirmation of expression of authentic
PHA synthase was obtained by Western analysis which also revealed the presence of several apparent proteolytic cleavage products. N-terminal sequence data were obtained from the 64-kDa
protein which verified its identity. The
PHA synthase produced in this system constitutes approximately 50% of total
protein after 60 h of
viral infection and is found approximately equally distributed in both soluble and membrane-associated fractions. The expression level allowed rapid purification of the soluble form of
PHA synthase to approximately 90% homogeneity in a single liquid chromatography step on
hydroxylapatite. Using a direct spectrophotometric assay, analyses show that the
enzyme has a pH optimum of 8.5, exhibits a concave-up Lineweaver-Burk plot, and a correlation between
enzyme concentration and specific activity. Over 1000 units of soluble
enzyme were obtained from a 250-ml culture of T. ni cells with an apparent initial specific activity of 12 mumol min-1 mg-1. The amount of
PHA synthase activity is significantly higher than previously obtained from much larger bacterial cultures. The method described here should provide a general approach for the expression of active PHA synthases from a variety of bacterial sources to facilitate substrate specificity and mechanistic studies of these intriguing
proteins.