FIG. 4.

Analysis of PHA-synthetic enzymes and granule-associated GA16 protein in recombinants of E. coli and P. denitrificans. E. coli cells were cultivated at 37°C in LB medium with ampicillin and kanamycin. (A) Total cellular proteins (20 μg per lane) were subjected to electrophoresis in an SDS–12.5% polyacrylamide gel and stained with CBB. Western blot analyses with anti-GA16 protein antibody (B), anti-β-galactosidase-PHA synthase fusion protein antibody (C), anti-β-ketothiolase antibody (D), and anti-acetoacetyl-CoA reductase antibody (E) are shown. Total cell proteins used for Western blot analysis with anti-GA16 protein antibody, anti-β-galactosidase-PHA synthase fusion protein antibody, anti-β-ketothiolase antibody, and anti-acetoacetyl-CoA reductase antibody were 4, 20, 10, and 10 μg per lane, respectively. The triangles in panels B, C, D, and E indicate GA16 protein, PHA synthase, β-ketothiolase, and acetoacetyl-CoA reductase, respectively. Lanes: 1, molecular mass standard proteins (same as for Fig. 1); 2, total cellular proteins of E. coli XL1-Blue (pBBRKmAB plus pTV119N); 3, total cellular proteins of E. coli XL1-Blue (pBBRKmAB plus pTVC); 4, total cellular proteins of E. coli XL1-Blue (pBBRKmAB plus pTVCP); 5, total cellular proteins of E. coli XL1-Blue (pBBRKmAB plus pTVCP4); 6, total cellular proteins of P. denitrificans; P, prestained SDS-PAGE standards (from top to bottom, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme; mobilities are not exact).