PURIFICATION AND CHARACTERIZATION OF A 14-KILODALTON PROTEIN THAT IS BOUND TO THE SURFACE OF POLYHYDROXYALKANOIC ACID GRANULES IN RHODOCOCCUS RUBER

The N-terminal amino acid sequence of the polyhydroxyalkanoic acid (PH A) granule-associated M(r)-15,500 protein of Rhodococcus ruber (the GA 14 protein) was analyzed. The sequence revealed that the corresponding structural gene is represented by open reading frame 3, encoding a pr otein with a calculated M(r) of 14,175 which was recently localized do wnstream of the PHA synthase gene (U. Pieper and A. Steinbuchel, FEMS Microbiol. Lett. 96:73-80, 1992). A recombinant strain of Escherichia coli XL1-Blue carrying the hybrid plasmid (pSKXA10) with open reading frame 3 overexpressed the GA14 protein. The GA14 protein was subseque ntly purified in a three-step procedure including chromatography on DE AE-Sephacel, phenyl-Sepharose CL-4B, and Superose 12. Determination of the molecular weight by gel filtration as well as electron microscopi c studies indicates that a tetrameric structure of the recombinant, na tive GA14 protein is most likely. Immunoelectron microscopy demonstrat ed 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 X. ruber indicated that expressi on of the GA14 protein depended strongly on PKA synthesis.