EXPRESSION AND ANALYSIS OF A BACTERIAL POLY(HYDROXYALKANOATE) SYNTHASE IN INSECT CELLS USING A BACULOVIRUS SYSTEM

An improved method for expression of poly-P-hydroxyalkanoate (PHA) syn thase from Alcaligenes eutrophus has been developed using the baculovi rus 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 sy nthase was obtained by Western analysis which also revealed the presen ce of several apparent proteolytic cleavage products. N-terminal seque nce data were obtained fi om the 64-kDa protein which verified its ide ntity. The PHA synthase produced in this system constitutes approximat ely 50% of total protein after 60 h of viral infection and is found ap proximately equally distributed in both soluble and membrane-associate d fractions. The expression level allowed rapid purification of the so luble form of PHA synthase to approximate to 90% homogeneity in a sing le liquid chromatography step on hydroxylapatite. Using a direct spect rophotometric assay, analyses show that the enzyme has a pH optimum of 8.5, exhibits a concave-up Lineweaver-Burk plot, and a correlation be tween enzyme concentration and specific activity. Over 1000 units of s oluble enzyme were obtained from a 250-ml culture of T. ni cells with an apparent initial specific activity of 12 mu mol min(-1) mg(-1).PHA synthase activity is significantly higher than previously obtained, fr om much larger bacterial cultures. The method described here should pr ovide a general approach for the expression of active PHA synthases fr om a variety of bacterial sources to facilitate substrate specificity and mechanistic studies of these intriguing proteins. (C) 1996 Academi c Press, Inc.