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The major outer membrane proteins Pgm6 (41 kDa) and Pgm7 (40 kDa) of Porphyromonas gingivalis ATCC 33277 are encoded by open reading frames pg0695 and pg0694, respectively, which form a single operon. Pgm6 and Pgm7 (Pgm6/7) have a high degree of similarity to Escherichia coli OmpA in the C-terminal region and are predicted to form eight-stranded [beta]-barrels in the N-terminal region. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pgm6/7 appear as bands with apparent molecular masses of 40 and 120 kDa, with and without a reducing agent, suggesting a monomer and trimer, respectively. To verify the predicted trimeric structure and function of Pgm6/7, we constructed three mutants with pg0695, pg0694, or both deleted. The double mutant produced no Pgm6/7. The single-deletion mutants appeared to contain less Pgm7 and Pgm6 and to form homotrimers that migrated slightly faster (115 kDa) and slower (130 kDa), respectively, than wild-type Pgm6/7 under nonreducing conditions. N-terminal amino acid sequencing and mass spectrometry analysis of partially digested Pgm6/7 detected only fragments from Pgm6 and Pgm7. Two-dimensional, diagonal electrophoresis and chemical cross-linking experiments with or without a reducing agent clearly showed that Pgm6/7 mainly form stable heterotrimers via intermolecular disulfide bonds. Furthermore, growth retardation and arrest of the three mutants and increased permeability of their outer membranes indicated that Pgm6/7 play an important role in outer membrane integrity. Based on results of liposome swelling experiments, these proteins are likely to function as a stabilizer of the cell wall rather than as a major porin in this organism.

BACKGROUND In the production of therapeutic proteins using Chinese hamster ovary ( CHO) cell culture, the compositional variability in raw materials is often poorly understood. To elucidate the effects of raw material variability in regulating the intracellular metabolic pathways, plant-derived supplements of wheat hydrolysates were employed as a model system of raw materials having high compositional variability. The intracellular metabolic fluxes of antibody-producing GS-CHO cells supplemented with different lots of wheat hydrolysates were quantified using the techniques of flux balance analysis ( FBA). RESULTS The examination of the metabolic flux clearly illustrated several unique metabolic features. Specifically, under the given environmental conditions induced by the variability of the different wheat hydrolysate lots, the serine and glycine metabolism could be linked to improved cell growth, while the elevated levels of the anaplerotic reaction and the TCA cycle could be related to the increased specific antibody production. Multivariate data analysis revealed the detailed metabolic regulations of a GS-CHO cell line in response to the compositional variability of the given raw materials. The study result can be used for targeted optimization of cell growth and monoclonal antibody production. CONCLUSION Metabolic flux analysis coupled with multivariate data analytics unravel the complicated effects of raw material variability on the cellular metabolism, and hence improve the overall productivity of an antibody-producing CHO cell culture. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]