Adaptive dissolved oxygen control through the glycerol feeding in a recombinant Pichia pastoris cultivation in conditions of oxygen transfer limitation

In high cell density cultivation processes the productivity is frequently constrained by the bioreactor maximum oxygen transfer capacity. The productivity can often be increased by operating the process at low dissolved oxygen concentrations close to the limitation level. This may be accomplished with a closed-loop controller that regulates the dissolved oxygen concentration by manipulating the dominant carbon source feeding rate. In this work we study this control problem in a pilot 50l bioreactor with a high cell density recombinant P. pastoris cultivation in complex media. The study focuses on the design of accurate stable adaptive controllers, with guaranteed exponential convergence and its relation with the calibration of controller parameters. Two adaptive control strategies were tested in the pilot bioreactor: a model reference adaptive controller with a linear reference model and an integral feedback controller with adaptive gain. The latter alternative proved to be more robust to errors in the measurements of the off-gas composition. Concerning the instrumentation, algorithms were derived assuming that both the dissolved oxygen tension and off-gas composition are measured on-line, but also the case of only dissolved oxygen being measured is addressed. It was verified that the measurement of off-gas composition might not improve the controller performance due to measurement and process time delays.     

Structural evidence for a proton transfer pathway coupled with haem reduction of cytochrome c″ from Methylophilus methylotrophus

The crystal structures of the oxidized and reduced forms of cytochrome c″ from Methylophilus methylotrophus were solved from X-ray synchrotron data to atomic resolution. The overall fold of the molecule in the two redox states is very similar and is comparable to that of the oxygen-binding protein from the purple phototrophic bacterium Rhodobacter sphaeroides. However, significant modifications occur near the haem group, in particular the detachment from axial binding of His95 observed upon reduction as well as the adoption of different conformations of some protonatable residues that form a possible proton path from the haem pocket to the protein surface. These changes are associated with the previously well characterized redox-Bohr behaviour of this protein. Furthermore they provide a model for one of the presently proposed mechanisms of proton translocation in the much more complex protein cytochrome c oxidase.     

Retrovirus producer cell line metabolism: implications on viral productivity

The production of retroviral vectors by human cell lines is still hampered by low titers making it relatively difficult to produce very large quantities of this vector of high interest for clinical gene therapy applications. Thus, to improve vector production, we studied the influence of different sugars alone or combinations of sugars on cell growth, vector titers, and metabolism of the producer cell. The use of fructose at 140 mM or a mixed medium (with glucose at 25 mM and fructose at 140 mM) improved the virus titer three- to fourfold, respectively, and the producer cell productivity by fivefold. The increase in the cell productivity was due to a 1.5-fold increase in the vector stability, the remaining increase being due to higher cell specific productivity. The increase in the productivity was associated with lower glucose oxidation and an increase in the lactate and alanine yield. In the mixed medium, an increase in fatty acids derived from the glucose was observed in parallel with a reduction of glutamate and glutamine synthesis via the tricarboxylic acid (TCA) cycle acetyl-CoA and α-ketoglutarate, respectively. Although the higher productivities were associated with severe changes in the glycolysis, TCA cycle, and glutaminolysis, the cell energetic status monitored by phosphocreatine and adenosine triphosphate levels was not significantly affected. The synthesis of fatty acids and phospholipids were enhanced in the fructose or mixed media and are possibly key parameters in retroviral vector production.     

Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability

The use of Moloney murine leukaemia virus (MoMLV) derived retroviral vectors in gene therapy requires the production of high titer preparations. However, obtaining high titers of infective MoMLV retroviral vectors is difficult due to the vector inherent instability. In this work the effect of the cell culture medium osmotic pressure upon the virus stability was studied. The osmolality of standard medium was raised from 335 up to 500 mOsm/kg using either ionic (sodium chloride) or non-ionic osmotic agents (sorbitol and fructose). It was observed that, independently of the osmotic agent used, the infectious vector inactivation rate was inversely correlated with the osmolality used in the production media; therefore, the use of high medium osmolalities enhanced vector stability. For production purposes a balance must be struck between cell yield, cell productivity and retroviral stability. From the conditions tested herein sorbitol addition, ensuring osmolalities between 410 and 450 mOsm/kg, yields the best production conditions; NaCl hampered the viral infectious production while fructose originates lower cell yields. Lipid extractions were performed for cholesterol and phospholipid analyses showing that more stable viral vectors had a 10% reduction in the cholesterol content. A similar reduction in cholesterol was observed in the producer cells. A detailed analysis of the major phospholipids composition, type and fatty acid content, by mass spectrometry did not show significant changes, confirming the decrease in the cholesterol to phospholipids ratio in the viral membrane as the major reason for the increased vector stability.     

Thermofluor-based optimization strategy for the stabilization and crystallization of Campylobacter jejuni desulforubrerythrin

Desulforubrerythrin from Campylobacter jejuni has recently been biochemical and spectroscopically characterized. It is a member of the rubrerythrin family, and it is composed of three structural domains: the N-terminal desulforedoxin domain with a non-heme iron center, followed by a four-helix bundle domain harboring a binuclear iron center and finally a C-terminal rubredoxin domain. To date, this is the first example of a protein presenting this kind of structural domain organization, and therefore the determination of its crystal structure may unveil unexpected structural features. Several attempts were made in order to obtain protein crystals, but always without success. As part of our strategy the thermofluor method was used to increase protein stability and its propensity to crystallize. This approach has been recently used to optimize protein buffer formulation, thus yielding more stable and homogenous protein samples. Thermofluor has also been used to identify cofactors/ligands or small molecules that may help stabilize native protein states. A successful thermofluor approach was used to select a pH buffer condition that allowed the crystallization of Campylobacter jejuni desulforubrerythrin, by screening both buffer pH and salt concentration. A buffer formulation was obtained which increased the protein melting temperature by 7°C relatively to the initial purification buffer. Desulforubrerythrin was seen to be stabilized by lower pH and high salt concentration, and was dialyzed into the new selected buffer, 100mM MES pH 6.2, 500mM NaCl. This stability study was complemented with a second thermofluor assay in which different additives were screened. A crystallization screening was carried out and protein crystals were rapidly obtained in one condition. Protein crystal optimization was done using the same additive screening. Interestingly, a correlation between the stability studies and crystallization experiments using the additive screening could be established. The work presented here shows an elegant example where thermofluor was shown to be a key biophysical method that allowed the identification of an improved buffer formulation and the applicability of this technique to increase the propensity of a protein to crystallize is discussed.     

Purification of recombinant rotavirus VP7 glycoprotein for the study of in vitro rotavirus-like particles assembly

Rotavirus VP7 is a glycoprotein that forms the viral capsid outerlayer and is essential to the correct assembly of triple-layered rotavirus-like particles (RLPs). In this work, a novel purification strategy was designed to allow obtaining highly pure monomeric VP7 required for the RLPs in vitro assembly. VP7 production kinetics in baculovirus-insect cells at cell concentration at infection (CCI) of 1x10(6)cellsmL(-1) was compared in terms of VP7/glycoprotein 64 (gp64) ratio at different multiplicity of infection (MOI). The best productivity was achieved at MOI of 0.1plaque forming unit (pfu)cell(-1) and time of harvest of 80h post-infection. After preliminary clarification steps, the proteins eluted from Concanavalin A were concentrated and loaded onto size exclusion chromatography. The polishing step was anion exchange chromatography with Mono Q. The high resolution of this column resulted in separation of monomers from dimers of VP7. Overall, the purification protocol yielded high level of purity (>90%). Purified VP7 was characterized by MALDI-TOF mass spectrometry and SDS-capillary gel electrophoresis. The MW and apparent MW were determined as 31.6 and 39kDa, respectively, confirming the efficacy of the proposed purification strategy that now enables RLPs assembly studies.     

[NiFe] hydrogenase from Desulfovibrio desulfuricans ATCC 27774: gene sequencing, three-dimensional structure determination and refinement at 1.8 Å and modelling studies of its interaction with the tetrahaem cytochrome c 3

The primary and three-dimensional structures of a [NiFe] hydrogenase isolated from D. desulfitricans ATCC 27774 were determined, by nucleotide analysis and single-crystal X-ray crystallography. The three-dimensional structural model was refined to R=0.167 and Rfree=0.223 using data to 1.8 A resolution. Two unique structural features are observed: the [4Fe-4S] cluster nearest the [NiFe] centre has been modified [4Fe-3S-3O] by loss of one sulfur atom and inclusion of three oxygen atoms; a three-fold disorder was observed for Cys536 which binds to the nickel atom in the [NiFe] centre. Also, the bridging sulfur atom that caps the active site was found to have partial occupancy, thus corresponding to a partly activated enzyme. These structural features may have biological relevance. In particular, the two less-populated rotamers of Cys536 may be involved in the activation process of the enzyme, as well as in the catalytic cycle. Molecular modelling studies were carried out on the interaction between this [NiFe] hydrogenase and its physiological partner, the tetrahaem cytochrome c3 from the same organism. The lowest energy docking solutions were found to correspond to an interaction between the haem IV region in tetrahaem cytochrome c3 with the distal [4Fe-4S] cluster in [NiFe] hydrogenase. This interaction should correspond to efficient electron transfer and be physiologically relevant, given the proximity of the two redox centres and the fact that electron transfer decay coupling calculations show high coupling values and a short electron transfer pathway. On the other hand, other docking solutions have been found that, despite showing low electron transfer efficiency, may give clues on possible proton transfer mechanisms between the two molecules.     

Crystal structure of low-potential cytochrome c 549 from Synechocystis sp. PCC 6803 at 1.21 Å resolution

The crystal structure of low-potential cytochrome c549, an extrinsic component of the photosystem II (PS II) from Synechocystis sp. PCC 6803, was obtained directly from single-wavelength 1.21 A resolution diffraction data. This is the first monodomain bis-histidinyl monoheme cytochrome c to be structurally characterized. The extended N-terminal region of c549 builds up a two-strand antiparallel beta-sheet in a hairpin motif, which extends through two molecules owing to crystal packing. Both peptide termini are involved in crystal contacts, which may explain their protrusion out of the globular fold. The C-terminus is preceded by a 9 A-long hydrophobic finger extending from a positively charged base and could be involved in PSII interactions, as well as a protruding negative patch built by a set of conserved acidic residues among c549 sequences.