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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.

Formulating appropriate storage conditions for biopharmaceutical proteins is essential for ensuring their stability and thereby their purity, potency, and safety over their shelf-life. Using a model murine IgG3 produced in a bioreactor system, multiple formulation compositions were systematically explored in a DoE design to optimize the stability of a challenging antibody formulation worst case. The stability of the antibody in each buffer formulation was assessed by UV/VIS absorbance at 280 nm and 410 nm and size exclusion high performance liquid chromatography (SEC) to determine overall solubility, opalescence, and aggregate formation, respectively. Upon preliminary testing, acetate was eliminated as a potential storage buffer due to significant visible precipitate formation. An additional 24 full factorial DoE was performed that combined the stabilizing effect of arginine with the buffering capacity of histidine. From this final DoE, an optimized formulation of 200 mM arginine, 50 mM histidine, and 100 mM NaCl at a pH of 6.5 was identified to substantially improve stability under long-term storage conditions and after multiple freeze/thaw cycles. Thus, our data highlights the power of DoE based formulation screening approaches even for challenging monoclonal antibody molecules. [ABSTRACT FROM AUTHOR]

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]

Integration host factor (IHF) is a DNA–bending protein that recognizes its cognate sites through indirect readout. Previous studies have shown that binding of wild-type (WT)-IHF is disrupted by a T to A mutation at the center position of a conserved TTR motif in its binding site, and that substitution of βGlu44 with Ala prevented IHF from discriminating between A and T at this position. We have determined the crystal structures and relative binding affinities for all combinations of WT-IHF and IHF-βGlu44Ala bound to the WT and mutant DNAs. Comparison of these structures reveals that DNA twist plays a major role in DNA recognition by IHF, and that this geometric parameter is dependent on the dinucleotide step and not on the bound IHF variant. [Copyright &y& Elsevier]

The 41 kDa heat-modifiable OmpA-like protein from Bacteroides fragilis ATCC 25285 was purified and tryptic peptide sequences were used to design degenerate oligonucleotide primers which were then used in amplification and identification of the omp A gene. The omp A gene was cloned and sequenced (GenBank Accession Number AY028459). Sequence analysis revealed an open reading frame of 1188 bases. The deduced amino acid sequence (which contained the experimentally determined peptide sequences) has 395 amino acids; the mature protein consists of 376 amino acids, has a predicted MW of 41 448 and a theoretical pI of 7.95. It is preceded by 19 amino acids that correspond to elements typical of a signal peptide sequence, including the Lys-Lys sequence near the N-terminus followed by a hydrophobic region (LLALAGV). The C-terminus of B. fragilis OmpA is homologous to the highly characteristic OmpA signature domain (75% similarity with the OmpA consensus domain). Three-dimensional modeling of B. fragilis OmpA predicts an eight-stranded β-barrel; the predicted loops are in agreement with those predicted as hydrophilic maxima in hydrophobicity plots. B. fragilis OmpA was cloned into pET-27b(+) and expressed in E. coli BL21(DE3)pLysS. The recombinant B. fragilis OmpA was recovered from inclusion bodies, solubilized in 8 M urea and refolded in 4 mM Zwittergent 3-14. Recombinant, refolded B. fragilis OmpA was active in a liposome assay (swelling rate for arabinose was 0.495/5.6μg protein) and preliminary analysis indicated that channels of three sizes were formed in a black lipid bilayer assay. This is the first description of an ompA homolog in Bacteroides. [Copyright &y& Elsevier]

The high MW porin protein complex (Omp200, composed of Omp121 and Omp71) from Bacteroides fragilis ATCC 25285 was purified and tryptic peptide sequences were used to design degenerate oligonucleotide primers which were then used as a first step in amplification, identification and sequencing of the omp121 gene (GenBank Accession Number AF357210). Sequence analysis revealed an open reading frame of 3378 bases. The deduced amino acid sequence (which contained the experimentally determined peptide sequences) has 1125 or 1116 amino acids (depending on which start codon is used); the mature protein consists of 1096 amino acids, has a predicted MW of 121.4 and a theoretical pI of 6.32. It is preceded by a 29 or 18 amino acid signal peptide which includes a typical hydrophobic region near the N-terminus (VLVLVL). Hydropathy plots of the deduced amino acid sequence of B. fragilis Omp121 display striking similarity with those of Escherichia coli OmpC (a 16-stranded porin) and FepA (a 22-stranded ligand-gated transport protein). Three-dimensional modeling of B. fragilis Omp121 (based on 1D and 3D sequence profiles, coupled with secondary structure and solvation potential information) indicated that the closest homologues in terms in fold conservation were the E. coli 16-stranded porins (e.g. OsmA) and 22-stranded ligand gated transport proteins (e.g. FepA). The omp71 gene sequence was identified using the tryptic peptides to search the published Bacteroides genome data base. We found that omp71 is located immediately downstream of omp121 and confirmed this with PCR analysis. Omp71 has no known homologues but does share some characteristics with the Porphyromonas RagB antigen. [Copyright &y& Elsevier]

Highlights: [•] We mapped packaging cleavage sites for phage 80α and pathogenicity island SaPI1. [•] Packaging directed by 80α TerS initiates within the 80α terS coding sequence. [•] SaPI1 TerS-specific packaging initiates in an intergenic region. [•] The SaPI1 pac site determinants were mapped using a plasmid-based assay. [•] Deletion of the pac site from SaPI1 eliminates high frequency transduction. [ABSTRACT FROM AUTHOR]

The activity of faropenem, a new oral penem, was tested against 579 strains of anaerobic bacteria by using the NCCLS-approved reference method. Drugs tested included amoxicillin-clavulanate, cefoxitin, clindamycin, faropenem, imipenem, and metronidazole. Of the 176 strains of Bacteroides fragilis group isolates tested, two isolates had faropenem MICs of 64 micro g/ml and imipenem MICs of >32 micro g/ml. Faropenem had an MIC of 16 micro g/ml for an additional isolate of B. fragilis; this strain was sensitive to imipenem (MIC of 1 micro g/ml). Both faropenem and imipenem had MICs of < or=4 micro g/ml for all isolates of Bacteroides capillosus (10 isolates), Bacteroides splanchnicus (13 isolates), Bacteroides ureolyticus (11 isolates), Bilophila wadsworthia (11 isolates), Porphyromonas species (42 isolates), Prevotella species (78 isolates), Campylobacter species (25 isolates), Sutterella wadsworthensis (11 isolates), Fusobacterium nucleatum (19 isolates), Fusobacterium mortiferum/varium (20 isolates), and other Fusobacterium species (9 isolates). Faropenem and imipenem had MICs of 16 to 32 micro g/ml for two strains of Clostridium difficile; the MICs for all other strains of Clostridium tested (69 isolates) were < or =4 micro g/ml. Faropenem had MICs of 8 and 16 micro g/ml, respectively, for two strains of Peptostreptococcus anaerobius (MICs of imipenem were 2 micro g/ml). MICs were < or =4 micro g/ml for all other strains of gram-positive anaerobic cocci (53 isolates) and non-spore-forming gram-positive rods (28 isolates). Other results were as expected and reported in previous studies. No metronidazole resistance was seen in gram-negative anaerobes other than S. wadsworthensis (18% resistant); 63% of gram-positive non-spore-forming rods were resistant. Some degree of clindamycin resistance was seen in most of the groups tested.