Analysis of a new flavodiiron core structural arrangement in Flv1-ΔFlR protein from Synechocystis sp. PCC6803

Flavodiiron proteins (FDPs) play key roles in biological response mechanisms against oxygen and/or nitric oxide; in particular they are present in oxygenic phototrophs (including cyanobacteria and gymnosperms). Two conserved domains define the core of this family of proteins: a N-terminal metallo-β-lactamase-like domain followed by a C-terminal flavodoxin-like one, containing the catalytic diiron centre and a FMN cofactor, respectively. Members of the FDP family may present extra modules in the C-terminus, and were classified into several classes according to their distribution and composition. The cyanobacterium Synechocystis sp. PCC6803 contains four Class C FDPs (Flv1-4) that include at the C-terminus an additional NAD(P)H:flavin oxidoreductase (FlR) domain. Two of them (Flv3 and Flv4) have the canonical diiron ligands (Class C, Type 1), while the other two (Flv1 and Flv2) present different residues in that region (Class C, Type 2). Most phototrophs, either Bacterial or Eukaryal, contain at least two FDP genes, each encoding for one of those two types. Crystals of the Flv1 two core domains (Flv1-ΔFlR), without the C-terminal NAD(P)H:flavin oxidoreductase extension, were obtained and the structure was determined. Its pseudo diiron site contains non-canonical basic and neutral residues, and showed anion moieties, instead. The presented structure revealed for the first time the structure of the two-domain core of a Class C-Type 2 FDP.     

Conformational component in the coupled transfer of multiple electrons and protons in a monomeric tetraheme cytochrome

Cell metabolism relies on energy transduction usually performed by complex membrane-spanning proteins that couple different chemical processes, e.g. electron and proton transfer in proton-pumps. There is great interest in determining at the molecular level the structural details that control these energy transduction events, particularly those involving multiple electrons and protons, because tight control is required to avoid the production of dangerous reactive intermediates. Tetraheme cytochrome c(3) is a small soluble and monomeric protein that performs a central step in the bioenergetic metabolism of sulfate reducing bacteria, termed "proton-thrusting," linking the oxidation of molecular hydrogen with the reduction of sulfate. The mechano-chemical coupling involved in the transfer of multiple electrons and protons in cytochrome c(3) from Desulfovibrio desulfuricans ATCC 27774 is described using results derived from the microscopic thermodynamic characterization of the redox and acid-base centers involved, crystallographic studies in the oxidized and reduced states of the cytochrome, and theoretical studies of the redox and acid-base transitions. This proton-assisted two-electron step involves very small, localized structural changes that are sufficient to generate the complex network of functional cooperativities leading to energy transduction, while using molecular mechanisms distinct from those established for other Desulfovibrio sp. cytochromes from the same structural family.     

Virus-like particle production at low multiplicities of infection with the baculovirus insect cell system

The baculovirus insect cell expression system (BEVS) was used for the production of self-forming Porcine parvovirus-like particles (VLPs) in serum-free medium. A low multiplicity of infection (MOI) strategy was used to overcome an extra virus amplification step, undesirable in industrial production, and to minimize the virus passage effect. It was confirmed that the time of infection (TOI) and MOI are dependent variables. Higher cell densities were obtained at low MOIs, keeping a constant TOI; however, both volumetric and specific productivities were lower. In synchronous infection, at high MOI, the specific productivity decreased when the cells were infected in the late phase of growth. Product degradation due to cell lysis strongly influenced the optimal time of harvest (TOH). Time of harvest was found to be highly dependent on the MOI, and a direct relationship with the cell yield was obtained.Analysis of the culture medium reveals that glutamine depletion occurs in the late phase of the growth. Supplementation of glutamine to uninfected cell cultures resulted in an increased cell yield. Its addition to cultures infected in the middle phase of the growth curve was also able to restore the productivity levels, but addition to cells in their stationary phase caused no observable effect on product expression. The study clearly shows that for a specific TOI it is not obvious what the correct MOI should be to obtain the best volumetric productivity.     

Crystal structure of a bacterial endospore coat component. A laccase with enhanced thermostability properties

Endospores produced by the Gram-positive soil bacterium Bacillus subtilis are shielded by a proteinaceous coat formed by over 30 structural components, which self-assemble into a lamellar inner coat and a thicker striated electrodense outer coat. The 65-kDa CotA protein is an abundant component of the outer coat layer. CotA is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light. Here, we report the structure of CotA at 1.7-A resolution, as determined by x-ray crystallography. This is the first structure of an endospore coat component, and also the first structure of a bacterial laccase. The overall fold of CotA comprises three cupredoxin-like domains and includes one mononuclear and one trinuclear copper center. This arrangement is similar to that of other multicopper oxidases and most similar to that of the copper tolerance protein CueO of Escherichia coli. However, the three cupredoxin domains in CotA are further linked by external interdomain loops, which increase the packing level of the structure. We propose that these interdomain loops contribute to the remarkable thermostability of the enzyme, but our results suggest that additional factors are likely to play a role. Comparisons with the structure of other monomeric multicopper oxidases containing four copper atoms suggest that CotA may accept the largest substrates of any known laccase. Moreover, and unlike other laccases, CotA appears to have a flexible lidlike region close to the substrate-binding site that may mediate substrate accessibility. The implications of these findings for the properties of CotA, its assembly and the properties of the bacterial spore coat structure are discussed.     

Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells

The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.     

Large scale production and downstream processing of a recombinant porcine parvovirus vaccine

Porcine parvovirus (PPV) virus-like particles (VLPs) constitute a potential vaccine for prevention of parvovirus-induced reproductive failure in gilts. Here we report the development of a large scale (25 l) production process for PPV-VLPs with baculovirus-infected insect cells. A low multiplicity of infection (MOI) strategy was efficiently applied avoiding the use of an extra baculovirus expansion step. The optimal harvest time was defined at 120 h post-infection at the MOI used, with the cell concentration at infection being 1.5x10(6) cells/ml. An efficient purification scheme using centrifugation, precipitation and ultrafiltration/diafiltration as stepwise unit operations was developed. The global yield of the downstream process was 68%. Baculovirus inactivation with Triton X-100 was successfully integrated into the purification scheme without an increase in the number of process stages. Immunogenicity of the PPV-VLPs tested in guinea pigs was similar to highly purified reference material produced from cells cultured in the presence of serum-containing medium. These results indicate the feasibility of industrial scale production of PPV-VLPs in the baculovirus system, safety of the product, and the potency of the product for vaccine application.     

Structural evidence for ligand specificity in the binding domain of the human androgen receptor. Implications for pathogenic gene mutations

The crystal structures of the human androgen receptor (hAR) and human progesterone receptor ligand-binding domains in complex with the same ligand metribolone (R1881) have been determined. Both three-dimensional structures show the typical nuclear receptor fold. The change of two residues in the ligand-binding pocket between the human progesterone receptor and hAR is most likely the source for the specificity of R1881 to the hAR. The structural implications of the 14 known mutations in the ligand-binding pocket of the hAR ligand-binding domains associated with either prostate cancer or the partial or complete androgen receptor insensitivity syndrome were analyzed. The effects of most of these mutants could be explained on the basis of the crystal structure.     

Modeling retrovirus production for gene therapy. 2. Integrated optimization of bioreaction and downstream processing

In this work a model envisaging the integrated optimization of bioreaction and downstream processing is presented. This model extends the work presented in part 1 of this pair of papers by adding ultrafiltration to process optimization. The new operational parameters include ultrafiltration time, pressure, and stirring rate. For global optimization, the model uses as constraints the final product titer and quality to be achieved after downstream processing. This extended model was validated with the same system used in part 1, i.e., PA317 cells producing a recombinant retrovirus containing the LacZ gene as a marker in stirred tanks using porous supports. Optimization of the extended model led to the conclusion that bioreaction should have two steps, batch and perfusion, similar to what was found in part 1. Ultrafiltration in a stirred cell should be performed at low pressures and stirring rates to reduce the losses of infective retroviruses. Sensitivity analysis performed on the results of the integrated optimization showed that under optimal conditions the productivity is less sensitive to the parameters related to ultrafiltration than to those associated with bioreaction. These results were interpreted as reflecting the high yield of ultrafiltration (90%). The relevance of the model extension to perform integrated optimization was also demonstrated since a restriction in the specific ultrafiltration area in downstream processing conditioned perfusion duration and perfusion rate in bioreaction. This clearly indicates that overall process optimization cannot be achieved without integrated optimization.     

Metabolically optimised BHK cell fed-batch cultures

The aim of this work was the optimisation of a fed-batch culture by metabolic confinement of BHK21 cells producing an antibody/cytokine fusion protein with potential application in tumour-targeted therapy. Previous results showed that at very low nutrient concentrations, a metabolic shift towards more efficient metabolic pathways occurs. The application of those results in the optimisation of a fed-batch culture resulted in higher cell growth (0.020 vs. 0.016 h(-1)) and cell viability, higher maximum cell concentration (2.5 vs. 1.1x10(6) cell ml(-1)), longer culture span (17 versus nine days) and higher product titre (60% increase), in relation to batch culture. This was achieved by maintaining glucose at 0.3 mM and glutamine at 0.2 mM through the addition of a concentrated solution based on the estimations of future nutrient consumption and growth rates through off line measurements. The production of toxic metabolites such as lactate and ammonia was reduced, especially the lactate production, which was markedly decreased due to the metabolic confinement of the cells. In conclusion, it was possible to increase the final titre of the recombinant antibody/cytokine fusion protein by confining the metabolism of the cells to an energetically more efficient state.