Implementation of Novel Affinity Ligand for Lentiviral Vector Purification.

The use of viral vectors as therapeutic products for multiple applications such as vaccines, cancer treatment, or gene therapies, has been growing exponentially. Therefore, improved manufacturing processes are needed to cope with the high number of functional particles required for clinical trials and, eventually, commercialization. Affinity chromatography (AC) can be used to simplify purification processes and generate clinical-grade products with high titer and purity. However, one of the major challenges in the purification of Lentiviral vectors (LVs) using AC is to combine a highly specific ligand with a gentle elution condition assuring the preservation of vector biological activity. In this work, we report for the first time the implementation of an AC resin to specifically purify VSV-G pseudotyped LVs. After ligand screening, different critical process parameters were assessed and optimized. A dynamic capacity of 1 × 1011 total particles per mL of resin was determined and an average recovery yield of 45% was found for the small-scale purification process. The established AC robustness was confirmed by the performance of an intermediate scale providing an infectious particles yield of 54%, which demonstrates the scalability and reproducibility of the AC matrix. Overall, this work contributes to increasing downstream process efficiency by delivering a purification technology that enables high purity, scalability, and process intensification in a single step, contributing to time-to-market reduction.

Nanoencapsulation of Gla-Rich Protein (GRP) as a Novel Approach to Target Inflammation.

Chronic inflammation is a major driver of chronic inflammatory diseases (CIDs), with a tremendous impact worldwide. Besides its function as a pathological calcification inhibitor, vitamin K-dependent protein Gla-rich protein (GRP) was shown to act as an anti-inflammatory agent independently of its gamma-carboxylation status. Although GRP's therapeutic potential has been highlighted, its low solubility at physiological pH still constitutes a major challenge for its biomedical application. In this work, we produced fluorescein-labeled chitosan-tripolyphosphate nanoparticles containing non-carboxylated GRP (ucGRP) (FCNG) via ionotropic gelation, increasing its bioavailability, stability, and anti-inflammatory potential. The results indicate the nanosized nature of FCNG with PDI and a zeta potential suitable for biomedical applications. FCNG's anti-inflammatory activity was studied in macrophage-differentiated THP1 cells, and in primary vascular smooth muscle cells and chondrocytes, inflamed with LPS, TNFα and IL-1ß, respectively. In all these in vitro human cell systems, FCNG treatments resulted in increased intra and extracellular GRP levels, and decreased pro-inflammatory responses of target cells, by decreasing pro-inflammatory cytokines and inflammation mediators. These results suggest the retained anti-inflammatory bioactivity of ucGRP in FCNG, strengthening the potential use of ucGRP as an anti-inflammatory agent with a wide spectrum of application, and opening up perspectives for its therapeutic application in CIDs.

Biophysical characterization of laforin-carbohydrate interaction.

Laforin is a human dual-specificity phosphatase (DSP) involved in glycogen metabolism regulation containing a carbohydrate-binding module (CBM). Mutations in the gene coding for laforin are responsible for the development of Lafora disease, a progressive fatal myoclonus epilepsy with early onset, characterized by the intracellular deposition of abnormally branched, hyperphosphorylated insoluble glycogen-like polymers, called Lafora bodies. Despite the known importance of the CBM domain of laforin in the regulation of glycogen metabolism, the molecular mechanism of laforin-glycogen interaction is still poorly understood. Recently, the structure of laforin with bound maltohexaose was determined and despite the importance of such breakthrough, some molecular interaction details remained missing. We herein report a thorough biophysical characterization of laforin-carbohydrate interaction using soluble glycans. We demonstrated an increased preference of laforin for the interaction with glycans with higher order of polymerization and confirmed the importance of tryptophan residues for glycan interaction. Moreover, and in line with what has been described for other CBMs and lectins, our results confirmed that laforin-glycan interactions occur with a favourable enthalpic contribution counter-balanced by an unfavourable entropic contribution. The analysis of laforin-glycan interaction through the glycan side by saturation transfer difference (STD)-NMR has shown that the CBM-binding site can accommodate between 5 and 6 sugar units, which is in line with the recently obtained crystal structure of laforin. Overall, the work in the present study complements the structural characterization of laforin and sheds light on the molecular mechanism of laforin-glycan interaction, which is a pivotal requisite to understand the physiological and pathological roles of laforin.

A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation.

Protein aggregation into insoluble amyloid fibrils is the hallmark of several neurodegenerative diseases, chief among them Alzheimer's and Parkinson's. Although caused by different proteins, these pathologies share some basic molecular mechanisms with familial amyloidotic polyneuropathy (FAP), a rare hereditary neuropathy caused by amyloid formation and deposition by transthyretin (TTR) in the peripheral and autonomic nervous systems. Among the amyloidogenic TTR mutations known, V30M-TTR is the most common in FAP. TTR amyloidogenesis (ATTR) is triggered by tetramer dissociation, followed by partial unfolding and aggregation of the low conformational stability monomers formed. Thus, tetramer dissociation kinetics, monomer conformational stability and competition between refolding and aggregation pathways do play a critical role in ATTR. Here, we propose a new model to analyze the refolding kinetics of WT-TTR and V30M-TTR, showing that at pH and protein concentrations close to physiological, a two-step mechanism with a unimolecular first step followed by a second-order second step adjusts well to the experimental data. Interestingly, although sharing the same kinetic mechanism, V30M-TTR refolds at a much slower rate than WT-TTR, a feature that may favor the formation of transient species leading to kinetic partition into amyloidogenic pathways and, thus, significantly increasing the probability of amyloid formation in vivo.

The apoptogenic toxin AIP56 is a metalloprotease A-B toxin that cleaves NF-κb P65.

AIP56 (apoptosis-inducing protein of 56 kDa) is a major virulence factor of Photobacterium damselae piscicida (Phdp), a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys(39)-Glu(40) peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.

A look into amyloid formation by transthyretin: aggregation pathway and a novel kinetic model.

The aggregation of proteins into insoluble amyloid fibrils is the hallmark of many, highly debilitating, human pathologies such as Alzheimer's or Parkinson's disease. Transthyretin (TTR) is a homotetrameric protein implicated in several amyloidoses like Senile Systemic Amyloidosis (SSA), Familial Amyloid Polyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC), and the rare Central Nervous System selective Amyloidosis (CNSA). In this work, we have investigated the kinetics of TTR aggregation into amyloid fibrils produced by the addition of NaCl to acid-unfolded TTR monomers and we propose a mathematically simple kinetic mechanism to analyse the aggregation kinetics of TTR. We have conducted circular dichroism, intrinsic tryptophan fluorescence and thioflavin-T emission experiments to follow the conformational changes accompanying amyloid formation at different TTR concentrations. Kinetic traces were adjusted to a two-step model with the first step being second-order and the second being unimolecular. The molecular species present in the pathway of TTR oligomerization were characterized by size exclusion chromatography coupled to multi-angle light scattering and by transmission electron microscopy. The results show the transient accumulation of oligomers composed of 6 to 10 monomers in agreement with reports suggesting that these oligomers may be the causative agent of cell toxicity. The results obtained may prove to be useful in understanding the mode of action of different compounds in preventing fibril formation and, therefore, in designing new drugs against TTR amyloidosis.

Assessing Multi-Attribute Characterization of Enveloped and Non-Enveloped Viral Particles by Capillary Electrophoresis.

Virus-based biopharmaceutical products are used in clinical applications such as vaccines, gene therapy, and immunotherapy. However, their manufacturing remains a challenge, hampered by the lack of appropriate analytical tools for purification monitoring or characterization of the final product. This paper describes the implementation of a highly sensitive method, capillary electrophoresis (CE)-sodium dodecyl sulfate (SDS) combined with a laser-induced fluorescence (LIF) detector to monitor the impact of various bioprocess steps on the quality of different viral vectors. The fluorescence labelling procedure uses the (3-(2-furoyl) quinoline-2-carboxaldehyde dye, and the CE-SDS LIF method enables the evaluation of in-process besides final product samples. This method outperforms other analytical methods, such as SDS-polyacrylamide gel electrophoresis with Sypro Ruby staining, in terms of sensitivity, resolution, and high-throughput capability. Notably, this CE-SDS LIF method was also successfully implemented to characterize enveloped viruses such as Maraba virus and lentivirus, whose development as biopharmaceuticals is now restricted by the lack of suitable analytical tools. This method was also qualified for quantification of rAAV2 according to the International Council for Harmonisation guidelines. Overall, our work shows that CE-SDS LIF is a precise and sensitive analytical platform for in-process sample analysis and quantification of different virus-based targets, with a great potential for application in biomanufacturing.

Advances in Lentivirus Purification.

Lentiviral vectors (LVs) have been increasingly used as a tool for gene and cell therapies since they can stably integrate the genome in dividing and nondividing cells. LV production and purification processes have evolved substantially over the last decades. However, the increasing demands for higher quantities with more restrictive purity requirements are stimulating the development of novel materials and strategies to supply the market with LV in a cost-effective manner. A detailed review of each downstream process unit operation is performed, limitations, strengths, and potential outcomes being covered. Currently, the majority of large-scale LV manufacturing processes are still based on adherent cell culture, although it is known that the industry is migrating fast to suspension cultures. Regarding the purification strategy, it consists of batch chromatography and membrane technology. Nevertheless, new solutions are being created to improve the current production schemes and expand its clinical use.

The N-terminal half of the peroxisomal cycling receptor Pex5p is a natively unfolded domain.

Targeting of most newly synthesised peroxisomal matrix proteins to the organelle requires Pex5p, the so-called PTS1 receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with these proteins in the cytosol, transports them to the peroxisomal membrane and catalyses their translocation across the membrane. Presently, our knowledge on the structural details behind the interaction of Pex5p with the cargo proteins is reasonably complete. In contrast, information regarding the structure of the Pex5p N-terminal half (a region containing its peroxisomal targeting domain) is still limited. We have recently observed that the Stokes radius of this Pex5p domain is anomalously large, suggesting that this portion of the protein is either a structured elongated domain or that it adopts a low compactness conformation. Here, we address this issue using a combination of biophysical and biochemical approaches. Our results indicate that the N-terminal half of Pex5p is best described as a natively unfolded pre-molten globule-like domain. The implications of these findings on the mechanism of protein import into the peroxisome are discussed.

Effect of ethyleneoxide groups of anionic surfactants on lipase activity.

The use of enzymes in laundry and dish detergent products is growing. Such tendency implies dedicated studies to understand surfactant-enzyme interactions. The interactions between surfactants and enzymes and their impact on the catalytic efficiency represent a central problem and were here evaluated using circular dichroism, dynamic light scattering, and enzyme activity determinations. This work focuses on this key issue by evaluating the role of the ethyleneoxide (EO) groups of anionic surfactants on the structure and activity of a commercial lipase, and by focusing on the protein/surfactant interactions at a molecular level. The conformational changes and enzymatic activity of the protein were evaluated in the presence of sodium dodecyl sulfate (SDS also denoted as SLE0 S) and of sodium lauryl ether sulfate with two EO units (SLE2 S). The results strongly suggest that the presence of EO units in the surfactant polar headgroup determines the stability and the activity of the enzyme. While SDS promotes enzyme denaturation and consequent loss of activity, SLE2 S preserves the enzyme structure and activity. The data further highlights that the electrostatic interactions among the protein groups are changed by the presence of the adsorbed anionic surfactants being such absorption mainly driven by hydrophobic interactions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1276-1282, 2016.

Mycobacterium hassiacum recovers from nitrogen starvation with up-regulation of a novel glucosylglycerate hydrolase and depletion of the accumulated glucosylglycerate.

Some microorganisms accumulate glucosylglycerate (GG) during growth under nitrogen deprivation. However, the molecular mechanisms underlying the role of GG and the regulation of its levels in the nitrogen stress response are elusive. Since GG is required for biosynthesis of mycobacterial methylglucose lipopolysaccharides (MGLP) we examined the molecular mechanisms linking replenishment of assimilable nitrogen to nitrogen-starved M. hassiacum with depletion of GG accumulated during nitrogen deficiency. To probe the involvement of a newly identified glycoside hydrolase in GG depletion, we produced the mycobacterial enzyme recombinantly and confirmed the specific hydrolysis of GG (GG hydrolase, GgH) in vitro. We have also observed a pronounced up-regulation of GgH mRNA in response to the nitrogen shock, which positively correlates with GG depletion in vivo and growth stimulation, implicating GgH in the recovery process. Since GgH orthologs seem to be absent from most slowly-growing mycobacteria including M. tuberculosis, the disclosure of the GgH function allows reconfiguration of the MGLP pathway in rapidly-growing species and accommodation of this possible regulatory step. This new link between GG metabolism, MGLP biosynthesis and recovery from nitrogen stress furthers our knowledge on the mycobacterial strategies to endure a frequent stress faced in some environments and during long-term infection.

A serpin released by an entomopathogen impairs clot formation in insect defense system.

Steinernema carpocapsae is an entomopathogenic nematode widely used for the control of insect pests due to its virulence, which is mainly attributed to the ability the parasitic stage has to overcome insect defences. To identify the mechanisms underlying such a characteristic, we studied a novel serpin-like inhibitor (sc-srp-6) that was detected in a transcriptome analysis. Recombinant Sc-SRP-6 produced in Escherichia coli had a native fold of serpins belonging to the α-1-peptidase family and exhibited inhibitory activity against trypsin and α-chymotrypsin with Ki of 0.42 × 10(-7) M and 1.22 × 10(-7) M, respectively. Functional analysis revealed that Sc-SRP-6 inhibits insect digestive enzymes, thus preventing the hydrolysis of ingested particles. Moreover, Sc-SRP-6 impaired the formation of hard clots at the injury site, a major insect defence mechanism against invasive pathogens. Sc-SRP-6 does not prevent the formation of clot fibres and the activation of prophenoloxidases but impairs the incorporation of the melanin into the clot. Binding assays showed a complex formation between Sc-SRP-6 and three proteins in the hemolymph of lepidopteran required for clotting, apolipophorin, hexamerin and trypsin-like, although the catalytic inhibition occurred exclusively in trypsin-like. This data allowed the conclusion that Sc-SRP-6 promotes nematode virulence by inhibiting insect gut juices and by impairing immune clot reaction.

Thermal unfolding kinetics of ubiquitin in the microsecond-to-second time range probed by Tyr-59 fluorescence.

Thermal folding/unfolding kinetics of wild-type ubiquitin (wt-UBQ) was studied in a wide time range, from microseconds to seconds, by combining rapid-mixing T-jump and laser T-jump with fluorescence detection (MTJ-F and LTJ-F, respectively) to monitor the fluorescence changes of Tyr-59 located on the 310-helix. The kinetics occurs exclusively in the millisecond to second time range, and the decays are strictly single exponential. From global analysis of folding and unfolding decays, the kf and ku values were determined, without use of the equilibrium constant Ku. The activation enthalpy of folding is negative (DeltaHf(#)(Tm) = -10.8 kcal/mol), but the free energy of the transition state is substantially larger than that of the unfolded state (DeltaGf(#)(Tm) = 7.6 kcal/mol RTm). Thus, wt-UBQ behaves as a two-state folder, when folding is monitored by the fluorescence of Tyr-59. The observation of kinetics on the microsecond time scale, when folding is monitored by the disruption of hydrogen bonds between beta-strands, using nonlinear infrared spectroscopy of the amide I vibrations (LTJ-DVE) [Chung, H. S.; Tokmakoff, A. Proteins: Struct., Funct., Bioinf. 2008, 72, 474-487], seems to result from the fact that MTJ-F monitors the effective unfolding (backbone exposure to water) of the thermally excited protein alone, while LTJ-DVE also monitors preliminary events (hydrogen-bond breaking) and thermal re-equilibration of the thermally excited protein.

FH8--a small EF-hand protein from Fasciola hepatica.

Vaccine and drug development for fasciolasis rely on a thorough understanding of the mechanisms involved in parasite-host interactions. FH8 is an 8 kDa protein secreted by the parasite Fasciola hepatica in the early stages of infection. Sequence analysis revealed that FH8 has two EF-hand Ca(2+)-binding motifs, and our experimental data show that the protein binds Ca(2+) and that this induces conformational alterations, thus causing it to behave like a sensor protein. Moreover, FH8 displays low affinity for Ca(2+) (K(obs) = 10(4) m(-1)) and is highly stable in its apo and Ca(2+)-loaded states. Homology models were built for FH8 in both states. It has only one globular domain, with two binding sites and appropriate groups in the positions for coordination of the metal ions. However, an unusually high content of positively charged amino acids in one of the binding sites, when compared with the prototypical sensor proteins, potentially affects the protein's affinity for Ca(2+). The only Cys present in FH8, conserved in the homologous proteins of other helminth parasites, is located on the surface, allowing the formation of dimers, detected on SDS gels. These findings reflect specificities of FH8, which are most probably related to its roles both in the parasite and in the host.

Biological activity of heterologous murine interleukin-10 and preliminary studies on the use of a dextrin nanogel as a delivery system.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which active form is a non-covalent homodimer with two intramolecular disulphide bonds essential for its biological activity. A mutated form of murine IL-10 was successfully expressed in E. coli, recovered and purified from inclusion bodies. Its ability to reduce tumor necrosis factor α synthesis and down-regulate class II major histocompatibility complex molecules expression on endotoxin-stimulated bone marrow-derived macrophages was confirmed, and shown to be similar to that of a commercially available IL-10. Given the potential of IL-10 for application in various medical conditions, it is essential to develop systems that can effectively deliver the protein. In this work it is shown that a dextrin nanogel effectively incorporate IL-10, stabilize, and enable the slow release of biologically active IL-10 over time. Altogether, these results demonstrate the suitability of dextrin nanogel to be used as a system for the controlled release of IL-10.

Enhancing the fluorescence of tyr-59 in ubiquitin by blocking proton transfer.

Two highly fluorescent mutants of ubiquitin (E51Q and E51A) were produced by mutation of a single amino acid, demonstrating that excited-state proton transfer from the tyrosine residue to the carboxylate group of Glu-51 in ubiquitin is responsible for the reduced fluorescence of wild-type ubiquitin (wt-UBQ) at pH 5. E51A shows a Tm=59 degrees C at pH 1.5 and a Tm>80 degrees C at pH 5 similar to wt-UBQ, which shows that the mutation has not altered the protein structure significantly. The high and constant fluorescence from pH 1.5 to pH 7 allows for the study of the folding/unfolding over a wide range of pHs which would otherwise be impossible with wt-UBQ.

Design of new enzyme stabilizers inspired by glycosides of hyperthermophilic microorganisms.

In response to stressful conditions like supra-optimal salinity in the growth medium or temperature, many microorganisms accumulate low-molecular-mass organic compounds known as compatible solutes. In contrast with mesophiles that accumulate neutral or zwitterionic compounds, the solutes of hyperthermophiles are typically negatively charged. (2R)-2-(alpha-D-Mannopyranosyl)glycerate (herein abbreviated as mannosylglycerate) is one of the most widespread solutes among thermophilic and hyperthermophilic prokaryotes. In this work, several molecules chemically related to mannosylglycerate were synthesized, namely (2S)-2-(1-O-alpha-D-mannopyranosyl)propionate, 2-(1-O-alpha-D-mannopyranosyl)acetate, (2R)-2-(1-O-alpha-D-glucopyranosyl)glycerate and 1-O-(2-glyceryl)-alpha-D-mannopyranoside. The effectiveness of the newly synthesized compounds for the protection of model enzymes against heat-induced denaturation, aggregation and inactivation was evaluated, using differential scanning calorimetry, light scattering and measurements of residual activity. For comparison, the protection induced by natural compatible solutes, either neutral (e.g., trehalose, glycerol, ectoine) or negatively charged (di-myo-inositol-1,3'-phosphate and diglycerol phosphate), was assessed. Phosphate, sulfate, acetate and KCl were also included in the assays to rank the solutes and new compounds in the Hofmeister series. The data demonstrate the superiority of charged organic solutes as thermo-stabilizers of enzymes and strongly support the view that the extent of protein stabilization rendered by those solutes depends clearly on the specific solute/enzyme examined. The relevance of these findings to our knowledge on the mode of action of charged solutes is discussed.

Protein stabilization by osmolytes from hyperthermophiles: effect of mannosylglycerate on the thermal unfolding of recombinant nuclease a from Staphylococcus aureus studied by picosecond time-resolved fluorescence and calorimetry.

2-O-alpha-Mannosylglycerate, a negatively charged osmolyte widely distributed among (hyper)thermophilic microorganisms, is known to provide notable protection to proteins against thermal denaturation. To study the mechanism responsible for protein stabilization, pico-second time-resolved fluorescence spectroscopy was used to characterize the thermal unfolding of a model protein, Staphylococcus aureus recombinant nuclease A (SNase), in the presence or absence of mannosylglycerate. The fluorescence decay times are signatures of the protein state, and the pre-exponential coefficients are used to evaluate the molar fractions of the folded and unfolded states. Hence, direct determination of equilibrium constants of unfolding from molar fractions was carried out. Van't Hoff plots of the equilibrium constants provided reliable thermodynamic data for SNase unfolding. Differential scanning calorimetry was used to validate this thermodynamic analysis. The presence of 0.5 m potassium mannosylglycerate caused an increase of 7 degrees C in the SNase melting temperature and a 2-fold increase in the unfolding heat capacity. Despite the considerable degree of stabilization rendered by this solute, the nature and population of protein states along unfolding were not altered in the presence of mannosylglycerate, denoting that the unfolding pathway of SNase was unaffected. The stabilization of SNase by mannosylglycerate arises from decreased unfolding entropy up to 65 degrees C and from an enthalpy increase above this temperature. In molecular terms, stabilization is interpreted as resulting from destabilization of the denatured state caused by preferential exclusion of the solute from the protein hydration shell upon unfolding, and stabilization of the native state by specific interactions. The physiological significance of charged solutes in hyperthermophiles is discussed.

Protein stabilisation by compatible solutes: effect of mannosylglycerate on unfolding thermodynamics and activity of ribonuclease A.

Differential scanning calorimetry, optical spectroscopy, and activity measurements were used to investigate the effect of mannosylglycerate, a negatively charged osmolyte widely distributed among thermophilic and hyperthermophilic archaea and bacteria, on the thermal unfolding of ribonuclease A (RNase A). For comparison, assays in the presence of trehalose, a canonical solute in mesophiles, and potassium chloride were also carried out. A thermodynamic analysis was performed by using differential scanning calorimetry data. The changes in the heat capacity for unfolding were similar for the different solutes examined. Mannosylglycerate was an efficient thermostabiliser of RNase A and induced an increase of 6 degrees C mole(-1) in the melting temperature. Moreover, the performance of mannosylglycerate as a stabiliser depended on the net charge of the molecule, with the maximal effect being observed at pH values above 4.5. Analysis of the enthalpic and entropic contributions to unfolding, derived from calorimetric data, revealed that the stabilisation rendered by mannosylglycerate is primarily achieved through a decrease in the unfolding entropy. Also, the number of protons taken up by RNase A upon denaturation in the presence of mannosylglycerate was considerably higher than with other solutes, a result consistent with a more rigid structure of the native protein. Mannosylglycerate (potassium salt) inhibited the activity of RNase A, albeit to a smaller extent than KCl, and acted as an efficient suppressor of aggregation of the denatured protein, thereby having a remarkable beneficial effect on the inactivation of RNase A upon thermal denaturation. The results are discussed in view of the physiological role of this charged compatible solute.