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Titolo:
EXPRESSION AND ANALYSIS OF A BACTERIAL POLY(HYDROXYALKANOATE) SYNTHASE IN INSECT CELLS USING A BACULOVIRUS SYSTEM
Autore:
WILLIAMS MD; FIENO AM; GRANT RA; SHERMAN DH;
Indirizzi:
UNIV MINNESOTA,BIOL PROCESS TECHNOL INST,240 GORTNER LABS,1479 GORTNER AVE ST PAUL MN 55108 UNIV MINNESOTA,BIOL PROCESS TECHNOL INST ST PAUL MN 55108 UNIV MINNESOTA,DEPT MICROBIOL ST PAUL MN 55108 PROCTER & GAMBLE CO,MIAMI VALLEY LABS,CORP RES DIV CINCINNATI OH 45223
Titolo Testata:
Protein expression and purification
fascicolo: 2, volume: 7, anno: 1996,
pagine: 203 - 211
SICI:
1046-5928(1996)7:2<203:EAAOAB>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
POLY-BETA-HYDROXYBUTYRATE; ALCALIGENES-EUTROPHUS H16; FATTY-ACID SYNTHASE; ESCHERICHIA-COLI; ZOOGLOEA-RAMIGERA; PHB BIOSYNTHESIS; GENE; THIOESTERASE; THIOLASE; CLONING;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
37
Recensione:
Indirizzi per estratti:
Citazione:
M.D. Williams et al., "EXPRESSION AND ANALYSIS OF A BACTERIAL POLY(HYDROXYALKANOATE) SYNTHASE IN INSECT CELLS USING A BACULOVIRUS SYSTEM", Protein expression and purification, 7(2), 1996, pp. 203-211

Abstract

An improved method for expression of poly-P-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 productionof 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 fi om 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 approximate to 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 withan apparent initial specific activity of 12 mu mol min(-1) mg(-1).PHAsynthase 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 specificityand mechanistic studies of these intriguing proteins. (C) 1996 Academic Press, Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 27/11/20 alle ore 02:16:08