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Hsp12 is a small heat shock protein of Saccharomyces cerevisiae upregulated in response to various stresses. Non recombinant Hsp12 has been purified and characterized. Using circular dichroism (CD), Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC), it has been demonstrated that the native Hsp12 is monomeric and intrinsically disordered (IDP). Hsp12 gains in structure in the presence of specific lipids (PiP(2)). The helical form binds to liposomes models membrane with high affinity, leading to their rigidification. These results suggest that hydrophobic and ionic interactions are involved. Hsp12 is most likely a membrane chaperone expressed during stresses in Saccharomyces cerevisiae.

Hsp12 is a small heat shock protein of Saccharomyces cerevisiae upregulated in response to various stresses. Non recombinant Hsp12 has been purified and characterized. Using circular dichroism (CD), Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC), it has been demonstrated that the native Hsp12 is monomeric and intrinsically disordered (IDP). Hsp12 gains in structure in the presence of specific lipids (PiP

High-throughput process development is more and more used in chromatography. Limitations are the tools provided by the manufacturers. Here, we describe a method to select ion exchange chromatographic media using a 96-well filter microplate.

Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure, thereby allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we independently evaluated cation exchangers to facilitate media selection and investigated the relationship between surface modification and chromatographic performances. Structure-extended resins showed higher binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained high capacities even with high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions with minimal residence/contact time. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity, and could contribute to cost reduction. In this work, we evaluated the dynamic binding capacities of various new ion exchange resins at different binding conductivities for different residence times, and observed that.

Mixed mode chromatography resins with salt tolerance, large design space and orthogonal selectivity requires a slightly more complex development than traditional resins. It is important to screen several ligands and several binding and elution conditions. This allows taking full advantage of these resins. High-Throughput Screening (HTS) for Process Development should be done with the help of Design of Experiment (DoE). It could be performed in filter plates or Robocolumns, and assisted by liquid handling automated workstation. Modeling of the results allows the choice of optimal parameters that can then be validated and scaled up. All this leads to a better knowledge and robustness of the purification step.

Mixed mode or multimodal chromatography has been developed for rational use of multiple interactions in a controlled manner, in contrast to non-specific interactions. Indeed, as the term "mixed mode" suggests, these resins allow different types of interactions within a single chromatographic medium. In this paper, HEA HyperCel™, PPA HyperCel™ mixed-mode chromatographic media have been studied. These mixed-mode sorbents typically involve hydrophobic pseudo-affinity interactions for binding and essentially ionic interactions (charge repulsion) for elution. We identified and characterized these different interactions in chromatographic experiments by exploiting specific properties of proteins using protein standards and complex mixtures. We highlighted the major intervention of at least two types of interactions in these media: hydrophobic and electrostatic interactions. We observed the behaviour of these resins at different pH, ionic strength, with different salts and buffers types and in the presence of different organic compounds.

Mixed mode chromatography offers a diversity of ligands, each providing a new selectivity. This allows the design of novel purification processes with reduced column steps. Structure of ligands is based on both hydrophobic and ionic groups. Thanks to its salt tolerance, crude extracts or post-IEX samples can be loaded directly without conditioning. The selectivity could be enhanced by modulating elution parameters or by using additives. More importantly, mixed mode chromatography could be as effective as affinity chromatography for mAb purification processes. Mixed mode chromatography opens the way to short and economical processes.

International audience; Hsp12 is a small heat shock protein produced in many organisms, including the yeast Saccharomyces cerevisiae. It has been described as an indicator of yeast stress rate and has also been linked to the sweetness sensation of wine. To obtain a sufficient amount of protein, we produced and purified Hsp12 without tag in Escherichia coli. A simple fast two-step process was developed using a microplate approach and a design of experiments. A capture step on an anion-exchange salt-tolerant resin was followed by size exclusion chromatography for polishing, leading to a purity of 97%. Thereafter, specific anti-Hsp12 antibodies were obtained by rabbit immunization. An ELISA was developed to quantify Hsp12 in various strains of Saccharomyces cerevisiae. The antibodies showed high specificity and allowed the quantitation of Hsp12 in the yeast. The quantities of Hsp12 measured in the strains differed in direct proportion to the level of expression found in previous studies.

Mixed mode chromatography, or multimodal chromatography, involves the exploitation of combinations of several interactions in a controlled manner, to facilitate the rapid capture of proteins. Mixed-mode ligands like HEA and PPA HyperCel™ facilitate different kinds of interactions (hydrophobic, ionic, etc.) under different conditions. In order to better characterize the nature of this multi-modal interaction, we sought to study a protein, lysozyme, which is normally not retained by these mixed mode resins under normal binding conditions. Lysozyme was modified specifically at Arginine residues by the action of phenylglyoxal, and was extensively studied in this work to better characterize the mixed-mode interactions of HEA HyperCel™ and PPA HyperCel™ chromatographic supports. We show here that the adsorption behaviour of lysozyme on HEA and PPA HyperCel™ mixed mode sorbents varies depending on the degree of charge modification at the surface of the protein. Experiments using conventional cation exchange and hydrophobic interaction chromatography confirm that both charge and hydrophobicity modification occurs at the surface of the protein after lysozyme reaction with phenylglyoxal. The results emanating from this work using HEA and PPA HyperCel sorbents strongly suggest that mixed mode chromatography can efficiently separate closely related proteins of only minor surface charge and/or hydrophobicity differences.

International audience; Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. We report herein the recombinant expression of three hydrophobic ELP5 (VPGIG)(n) with variable lengths (n = 20, 40, 60) and sub-ambient transition temperatures. These ELPs were purified from the cytoplasmic soluble fraction of Escherichia coli by inverse transition cycling, and their exact molecular weight was confirmed by various mass spectrometry techniques. Transition temperatures of ELP20, ELP40, and ELP60 were measured at 18.6 degrees C, 12.4 degrees C and 11.7 degrees C, respectively.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a 40-residue cationic peptide originally purified from human blood ultrafiltrate. The native peptide contains two disulfide bonds and is unique regarding its primary structure. Its biological role is not known but a previous study showed that chemically synthesized LEAP-2 exhibited in vitro antimicrobial activities against several Gram-positive bacteria. In order to determine its antimicrobial mode of action, we expressed human recombinant LEAP-2 in Escherichia coli. Circular dichroism spectroscopy and nuclear magnetic resonance analyses showed that the structure of the recombinant peptide was identical to that of the chemically synthesized and oxidized LEAP-2, with two disulfide bonds between Cys residues in relative 1-3 and 2-4 positions. Minimal inhibitory concentration (MIC) of the recombinant human LEAP-2 was determined by a conventional broth dilution assay. It was found to be bactericidal against Bacillus megaterium at a 200microM concentration. Interestingly, the linear LEAP-2 had a greater antimicrobial activity with a MIC value of 12.5microM, which was comparable to that of magainin2. SYTOX Green uptake was used to assess bacterial membrane integrity. Linear LEAP-2 and magainin2 permeabilized B. megaterium membranes with the same efficiency, whereas oxidized LEAP-2 did not induce stain uptake. Binding of the peptides to plasmid DNA was evaluated by gel retardation assays. The DNA-binding efficacy of linear LEAP-2 was three times higher than that of the peptide-containing disulfide bridges. Altogether, these results show that the secondary structure of human LEAP-2 has a profound impact on its antibacterial activity.

Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure and allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we suggested an independent evaluation of major anion exchangers to facilitate media selection, and investigated the relationship between (i) surface modification and (ii) chromatographic performances. Structure-extended resins showed higher binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained mainly high capacities even with extremely high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity and could contribute to cost reduction.

The development of a capture step of a human recombinant F(ab′)2 produced and expressed in baculovirus-infected cells was investigated by screening three mixed-mode chromatography sorbents (HEA HyperCel™, PPA HyperCel and MEP HyperCel) and two ion exchangers (Q Ceramic HyperD® F, S Ceramic HyperD F) sorbents using a 96-well plate format and SELDI-MS. HEA HyperCel gave the best separation performance therefore the conditions tested in micro-plate were transferred to laboratory scale chromatographic experiments, confirming that the recombinant F(ab′)2 was effectively captured on the mixed-mode sorbent without any pre-treatment of the crude extract with a 82% recovery and a 39-fold purification.

Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50-60% range for affinity resins, and in the 80-85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein.

Developments in packing and packing port design of radial columns in recent years have resulted in a claimed significant increase in performance of this process chromatography technology. In this first study, the main chromatographic parameters as efficiency, capacity factor, asymmetry and resolution were evaluated in a unique one-to-one comparison between a 120 ml bed-volume and 6 cm bed length radial chromatography mini-process column against a 50 mm diameter, 6 cm bed height and 120 ml bed-volume axial chromatography column. Radial chromatography showed an increase in efficiency by 31% in the number of plates per meter while the equilibration could be reduced by 0.4-0.5 column volumes. The asymmetry factor for bovine serum albumin in radial chromatography showed a reduction of 20% while the reduction of the asymmetry factor of the smaller protein ovotransferrin decreased even by 46% in comparison to the performance of the comparative axial chromatography column. Therefore in radial chromatography resolution improved up to 20%. The retention volume was similar in both cases. For radial chromatography, the decrease in "width at half height" at Height Equivalent of Theoretical Plates (HETP) measurements was 40% while the decrease of the over-all width of the peak was 27%. For adsorbed/desorbed proteins, the elution peak showed similar results: "width at half height" decreased to 45% while the over-all width of the peak decreased by 28%. The concentration of the non-retained protein in the flow-through (lysozyme), increased by 35% while the concentration of the eluted fraction (serum albumin bovine), increased with 40% in the radial chromatography columns. The better results obtained with the radial column were probably the consequence of the geometrical design of this device (larger inlet surface area and small outlet surface area which concentrate the eluted fraction).

International audience; An innovative process to purify mAb from CHO cell culture supernatant was developed. This three-step process involved two mixed mode resins and an anion exchange membrane. We used a human IgG mixture to determine the optimal conditions for each purification step. Thereafter, the whole process was evaluated and improved for the purification of a recombinant mAb produced in the supernatant of CHO cells. Once optimized, yield and purity of 88% and 99.9%, respectively were comparable to those obtained in a conventional process based on a capture step using protein A. In addition, aggregates, HCPs and DNA levels in the purified fraction were below regulatory specifications. Then we used mass spectrometry to identify contaminating proteins in the antibody fraction in order to highlight the behavior of HCPs. (C) 2015 Elsevier B.V. All rights reserved.

International audience; Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)(n) with variable lengths (n = 20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127 mg/L culture. After digestion of the fusion proteins by enterokinase, the ELF moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4 degrees C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELF of low molecular mass

5 pages; International audience; The enhanced green fluorescent protein (EGFP) was over-expressed in Escherichia coli as inclusion bodies to increase its quantity and to facilitate its purification. Insoluble EGFP has been purified on Q Hyper Z matrix by expanded bed adsorption after solubilization in 8M urea. The adsorption was made in expanded bed mode to avoid centrifugation. EBA-column refolding was done by elimination of urea and elution with NaCl. The EGFP was obtained as a highly purified soluble form with similar behavior in fluorescence and electrophoresis as native EGFP.

The aim of this work was to test a chromatographic support, 4-mercaptoethyl pyridine (4-MEP) Hypercel, for penicillin acylase purification by using pure penicillin acylase and crude extract. Two equilibration buffers with various salt concentrations and different flow rates were tested. The relationships between electrostatic and hydrophobic interactions and proteins are demonstrated. (NH4)2SO4 proved preferable because no salting-in occurred, contrary to NaCl. The recovery and purification fold were similar to those obtained in pseudo-affinity chromatography with a three-fold reduction of the (NH4)2SO4 concentration.

Three anion exchanger expanded bed adsorption (EBA) matrices: Streamline DEAE, Streamline Q XL and Q Hyper Z were evaluated with the aid of EFGP from an ultrasonic homogenate of Escherichia coli. Two pH of buffer were tested. Capture was done in an expanded mode whereas elution was done in a packed mode. The same conditions were chosen for evaluation of the three matrices. We observed a loss of EGFP (8-15%) in the through flow fraction especially with the Streamline Q XL matrix, probably due to an aggregation of beads during sample application. The beads of this matrix possess tentacles which probably retain a lot of cellular and molecular debris. The two other matrices gave a good purification of the EGFP (7-15-fold) but the Q Hyper Z matrix appeared to give the best results. It is composed of little size and density beads which lead to a higher exchange surface and then a better mass transfer.

The phytopathogenic fungus Botrytis cinerea produces a set of polygalacturonases (PGs) which are involved in the enzymatic degradation of pectin during plant tissue infection. Two polygalacturonases secreted by B. cinerea in seven-day-old liquid culture were purified to apparent homogeneity by chromatography. PG I was an exopolygalacturonase of molecular weight 65 kDa and pI 8.0 and PG II was an endopolygalacturonase of 52 kDa and pI 7.8. Enzymatic activity of PG I and PG II was partially inhibited by 1 mM CaCl2, probably by calcium chelation of polygalacturonic acid, the substrate of the enzyme.

International audience; Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long-sought hormone that regulates iron homeostasis in mammals. Recombinant human Hepcidin-25 (Hepc25) was expressed in Pichia pastoris using a modified version of the pPICZ alpha A vector. Hepc25 was then purified by a simple two-step chromatographic process to obtain 1.9 mg of soluble recombinant human Hepc25 per liter of culture at 96% purity. The sequence of Hepc25 and the presence of four disulfide bridges were confirmed by mass spectrometry analyses, and the recombinant Hepc25 exhibited antibacterial activity. This protocol of production and purification is the first step toward the production of human Hepc25 at a greater scale. (C) 2015 Elsevier B.V. All rights reserved.

Antimicrobial peptides (AMPs) are cationic molecules that are good leads for new antiinfective drugs. To obtain sufficient amounts, recombinant AMPs are generally produced as fusion proteins in Escherichia coli. Fusion partners facilitate purification of recombinant proteins. Fusion proteins are then cleaved by specific proteases, and cationic peptides are purified by size exclusion chromatography or ion exchange chromatography, neither of which is easily applicable to small volumes of diluted peptide samples. We developed a small-scale system that is easily adaptable for high-throughput screening and uses carboxyl magnetic beads to purify a cationic peptide from its fusion partner.

We evaluated mixed mode chromatography for the capture of recombinant antibodies from CHO cell culture supernatants. We studied PPA HyperCel, HEA HyperCel, MEP HyperCel and Capto adhere resins, which all contain hydrophobic and cationic groups. A microplate approach combined with DoE modeling allowed the exploration of the complex behaviors of these mixed mode resins. Optimal conditions for antibody purification and host cell proteins (HCPs) elimination were determined and then directly up-scaled to laboratory columns. Then we used mass spectrometry to identify the major HCPs potentially coeluted with the antibody. Differences between the four resins in terms of amount, complexity and identity of the HCPs present in the elution fractions were investigated.

International audience; Oenococcus oeni, the major lactic acid bacteria involved in malolactic fermentation (MLF) in wine, is able to produce volatile sulfur compounds from methionine. Methional reduction is the last enzymatic step of methionol synthesis in methionine catabolism. Alcohol dehydrogenase (ADH) activity was found to be present in the soluble fraction of O. oeni IOEB 8406. An NAD(P)H-dependent ADH involved in the reduction of methional was then purified to homogeneity. Sequencing of the purified enzyme and amino acid sequence comparison with the database revealed the presence of a conserved sequence motif specific to the medium-chain zinc-containing NAD(P)H-dependent ADHs. Despite the great importance of ADH activities in wine flavor modification, this is the first report of the purification of an ADH isolated from O. oeni. The purified ADH does not seem to be involved in the modification of buttery and lactic notes or to be involved in the specific formation of volatile alcohols during MLF. The enzyme was not strictly specific of methional reduction and the highest reducing activity was obtained with acetaldehyde as substrate. The function of the purified ADH remains unclear, although the role of the sulfur atom in methional molecules in the interaction between enzyme and substrate was evidenced.

Background The human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Results Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization. Conclusion We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

Hsp12 is a heat shock protein (12 kDa) expressed by the yeast Saccharomyces cerevisiae and associated with the stress response. Indeed, it has been shown that transcripts of the HSP12 gene are expressed in response to different stresses. In addition, the protein Hsp12 would be responsible for the sweetness of wine observed during the autolysis of yeasts during vinification. However, the sweet taste could come from the entire Hsp12, or from one or more peptides derived from Hsp12. The objective of this study was to obtain the pure native Hsp12 protein from Saccharomyces cerevisiae culture in order to understand its role, on the one hand in the stress response in yeast and on the other hand in the sweetness of wine.Cultures of the Saccharomyces cerevisiae Fx10 enological strain were made to study the native Hsp12 protein. The production of the Hsp12 protein in response to different stresses was studied during the cultures, thanks to an ELISA assay developed during this study. It has thus been demonstrated that the Hsp12 protein is produced in significantly greater quantities in response to thermal and osmotic stress. Ethanol stress causes a decrease in the amount of Hsp12 protein. The native Hsp12 protein extracted from the cultures was purified. A 3-step purification process has been developed. Several resins and chromatographic conditions were screened in microplates. PPA HyperCel mixed-mode resin has eliminated major contaminants due to its selectivity. Steric exclusion chromatography allowed the removal of remaining contaminants and thus obtain the native Hsp12 protein with a purity of 99%. Various biophysical and calorimetric techniques were used to characterize the purified native Hsp12 protein in the presence of model membranes. It has thus been demonstrated that the Hsp12 protein is an intrinsically disordered protein (IDP). It is characterized by the absence of secondary structures in aqueous solution and by the formation of α helices in the presence of SDS and phospholipid PiP2. The binding with PiP2 suggests a role in the stabilization of the plasma membrane of yeasts. The Hsp12 protein could thus act as a membrane chaperone. Organoleptic characterization of the purified native Hsp12 protein was also performed. It appears that the entire Hsp12 protein is not responsible for the sweetness but rather one or more peptides resulting from its enzymatic digestion.; La protéine Hsp12 est une protéine de choc thermique (12 kDa) exprimée par la levure Saccharomyces cerevisiae et associée à la réponse au stress. En effet, il a été montré que les transcrits du gène HSP12 sont exprimés en réponse à différents stress. De plus, la protéine Hsp12 serait responsable de la sucrosité du vin observée au cours de l’autolyse des levures lors de la vinification. Cependant, le goût sucré pourrait provenir de la protéine Hsp12 entière, ou, d’un ou plusieurs peptides issus de la protéine Hsp12. L’objectif de cette étude était d’obtenir la protéine Hsp12 native pure à partir de culture de Saccharomyces cerevisiae afin de comprendre son rôle, d’une part dans la réponse au stress chez la levure et, d’autre part dans la sucrosité du vin.Des cultures de la souche œnologique Fx10 de Saccharomyces cerevisiae ont été réalisées afin d’étudier la protéine Hsp12 native. La production de la protéine Hsp12 en réponse à différents stress a été étudiée au cours des cultures, grâce à un dosage ELISA développé lors de cette étude. Il a ainsi été mis en évidence que la protéine Hsp12 est produite en quantités significativement supérieures en réponse à des stress thermiques et osmotiques. Le stress éthanolique quant à lui entraine une diminution de la quantité de protéine Hsp12. La protéine Hsp12 native extraite à partir des cultures a été purifiée. Un procédé de purification en 3 étapes a été développé. Plusieurs résines et conditions chromatographiques ont été criblées en microplaques. La résine en mode mixte PPA HyperCel a permis d’éliminer des contaminants majeurs grâce à sa sélectivité. La chromatographie d’exclusion stérique a permis d’éliminer les contaminants restants et ainsi d’obtenir la protéine Hsp12 native avec une pureté de 99%. Différentes techniques biophysiques et calorimétriques ont permis de caractériser la protéine Hsp12 native purifiée, en présence de membranes modèles. Il a ainsi été démontré que la protéine Hsp12 est une protéine intrinsèquement non ordonnée (intrinsically disordered protein - IDP). Elle est caractérisée par l’absence de structures secondaires en solution aqueuse et par la formation d’hélices α en présence de SDS et du phospholipide PiP2. La liaison avec le PiP2 suggère un rôle dans la stabilisation de la membrane plasmique des levures. La protéine Hsp12 pourrait ainsi avoir un rôle de chaperonne de membrane. Une caractérisation organoleptique de la protéine Hsp12 native purifiée a également été réalisée. Il apparait que la protéine Hsp12 entière n’est pas responsable de la sucrosité mais plutôt un ou des peptides issus sa digestion enzymatique.