title: Expression and analysis of a bacterial poly(hydroxyalkanoate) synthase in insect cells using a baculovirus system.
abstract: 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.
authors.affiliation: Department of Microbiology and Biological Process Technology Institute, University of Minnesota, Saint Paul 55108-6106, USA.
authors.forename: M D
authors.initials: MD
authors.lastname: Williams
authors.forename: A M
authors.initials: AM
authors.lastname: Fieno
authors.forename: R A
authors.initials: RA
authors.lastname: Grant
authors.forename: D H
authors.initials: DH
authors.lastname: Sherman
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D000421:Alcaligenes / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007313:Insecta / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013053:Spectrophotometry
chemical_list: D000214:Acyl Coenzyme A; D010455:Peptides; D011994:Recombinant Proteins; C030372:3-hydroxybutyryl-coenzyme A; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase