Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Patent
1996-08-26
2000-02-08
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
43525233, 4353201, 435471, 435488, 536 237, C12N 121, C12N 1531, C12N 1570, C12N 1574
Patent
active
06022729&
ABSTRACT:
The N-terminal amino acid sequence of the polyhydroxyalkanoic acid (PHA) granule-associated M.sub.r 15,500 protein of Rhodococcus ruber, which is referred to as the GA14-protein, was analysed. The sequence revealed that the corresponding structural gene is represented by the open reading frame 3 encoding a protein with a calculated M.sub.r 14,175 which was recently localized downstream of the PHA synthase gene (Pieper, U., and A. Steinbuchel, 1992. FEMS Microbiol. Lett. 96: 73-80). A recombinant strain of Escherichia coli XL1-Blue carrying the hybrid plasmid (pSKXA10*) with ORF3 overexpressed the GA14-protein. The GA14-protein was subsequently purified in a three-step procedure including chromatography on DEAE-Sephacel, Phenyl-Sepharose CL-4B and Superose 12. Determination of the molecular weight by gel filtration as well as electron microscopic studies make a tetrameric structure of the recombinant, native GA14-protein most likely. Immunoelectron microscopy demonstrated a localization of the GA14-protein at the periphery of PHA granules as well as close to the cell membrane in R. ruber. Investigations of PHA-leaky and PHA-negative mutants of R. ruber indicated that the expression of the GA14-protein depended strongly on PhA synthesis.
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Pieper-Furst Ursula
Steinbuchel Alexander
Bond Gary M.
Monsanto Company
Nelson Amy J.
Robinson Douglas W.
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