1887

Abstract

In , the H16 phasin PhaP1 represents the major phasin that binds to the surface of polyhydroxyalkanoate (PHA) inclusions. In this study, C-terminal fusions of PhaP1 with enhanced green fluorescent protein (eGFP) and with -galactosidase (LacZ) were expressed separately in the triacylglycerol (TAG)-accumulating actinomycetes PD630 and mc155, employing the acetamidase () promoter of the / shuttle plasmid pJAM2. PhaP1 and the PhaP1 fusion proteins were expressed stably in the recombinant strains. Western blot analysis of cell fractions of revealed that PhaP1 and the PhaP1–eGFP fusion protein were associated with the TAG inclusions, whereas no phasin or phasin fusion protein was detected in the soluble and membrane fractions. Additional electron microscopy/immunocytochemistry studies demonstrated that PhaP1 was mainly located on the surface of intracellular TAG inclusions; in addition, some PhaP1 also occurred at the plasma membrane. Fluorescence microscopic investigations of the subcellular distribution of the PhaP1–eGFP fusion protein and on isolated TAG inclusions revealed that the fusion protein was bound to TAG inclusions at all stages of their formation, and to some extent at the cytoplasmic membrane. The PhaP1–LacZ fusion protein also bound to the TAG inclusions, and could be separated together with the inclusions from crude extracts, thus demonstrating the immobilization of -galactosidase activity on the inclusions. This is believed to be the first report demonstrating the ability of PhaP1 to bind to lipid inclusions in addition to PHA inclusions. Furthermore, it was demonstrated that this non-specificity of PhaP1 can be utilized to anchor enzymically active fusion proteins to a matrix of bacterial TAG inclusions.

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2006-11-01
2019-11-19
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