Production, in Pichia pastoris, of a recombinant monomeric mapacalcine, a protein with anti-ischemic properties.

Mapacalcine is a small homodimeric protein of 19 kDa with 9 disulfide bridges extracted from the Cliona vastifica sponge (Red Sea). It selectively blocks a calcium current insensitive to most calcium blockers. Specific receptors for mapacalcine have been described in a variety of tissues such as brain, smooth muscle, liver, and kidney. Previous works achieved on hepatocytes and nervous cells demonstrated that this protein selectively blocks a calcium influx triggered by an ischemia/reperfusion (I/R) shock and efficiently protects cells from death after I/R. The aim of this work was to produce the recombinant mapacalcine in the yeast Pichia pastoris. Mass spectrometry, light scattering analysis and biological characterization demonstrated that the recombinant mapacalcine obtained was a monomeric form with 4 disulfide bridges which retains the biological activity of the natural protein.

A comprehensive evaluation of mixed mode interactions of HEA and PPA HyperCel™ chromatographic media.

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.

A study on the nature of interactions of mixed-mode ligands HEA and PPA HyperCel using phenylglyoxal modified lysozyme.

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.

Cation exchange versus multimodal cation exchange resins for antibody capture from CHO supernatants: identification of contaminating host cell proteins by mass spectrometry.

We compared classical and multimodal cation exchange resins for the capture of recombinant antibodies from Chinese hamster ovary (CHO) cell culture supernatant. Both Capto S and Capto MMC resins present anionic groups while the multimodal Capto MMC also features a hydrophobic moiety. First we screened optimal binding and elution conditions in microplates with a pure antibody. We validated the results on the lab-scale with columns with a pure antibody and a CHO cell culture supernatant. Both resins achieved good yield and purity for the capture step of an antibody. However, the multimodal resin appeared more efficient and selective. Then we identified proteins in the antibody fraction by mass spectrometry in order to highlight the behavior of host cell proteins (HCPs).

Rapid screening of purification strategies for the capture of a human recombinant F(ab')2 expressed in baculovirus-infected cells using a micro-plate approach and SELDI-MS.

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.

Purification of an alcohol dehydrogenase involved in the conversion of methional to methionol in Oenococcus oeni IOEB 8406.

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.

Large-scale production, bacterial localization assessment and immobilized metal affinity chromatography purification of a human single-chain Fv antibody against alphaIIb-beta3 integrin.

Our objective was to investigate the Escherichia coli localization (such as supernatant, cytoplasm and inclusion bodies) of an anti-alphaIIb-beta3 (alphaIIbbeta3) scFv fragment referred to as scFv[EBB3] produced in batch fermentation. Immobilized metal affinity chromatography (IMAC) purification was performed on supernatant using expanded bed absorbed technology (EBA) and on sonicated cells in native conditions over an immobilized copper-ion affinity column. Inclusion bodies were solubilized before IMAC purification and the refolding procedure was performed on the column. The majority of scFv[EBB3] were present as inclusion bodies (55%), whereas 36% were found in the cytoplasm and only 9% secreted in the supernatant. The scFv activity was assessed by enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunohistochemistry analyses performed on a thrombus induced in vivo on an atherosclerotic rabbit model.

Purification and on-column refolding of EGFP overexpressed as inclusion bodies in Escherichia coli with expanded bed anion exchange chromatography.

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 8 M 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.

Improvement in production and purification bioprocesses of bacterially expressed anti-alphaIIbbeta3 human single-chain FV antibodies.

Production of anti-alphaIIbbeta3 (anti-alphaIIbbeta3)-binding single-chain FV (scFv) fragments obtained from combinatorial libraries of IgG human antibodies is of broad interest for imaging and treatment of acute coronary syndromes. The objective of our work was to design an optimized production of one selected anti-alphaIIbbeta3-binding scFv fragment for subsequent in vivo animal studies. Fed-batch fermentation was initiated with 2TY media supplemented with 0.1 M glucose. This growing batch culture was used as a starting point for further fed-batch induction, in which a media without glucose containing 1 mM IPTG and 0.4 M saccharose was continuously added. Subsequent purification was performed on the whole cell extract in native conditions over an immobilized copper-ion affinity column. The improved conditions allowed the recovery of 5 mg of highly purified scFv fragments as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bioactivity of the scFv fragments was further monitored by ELISA, cytometric and immunohistochemical methods.

Evaluation of three expanded bed adsorption anion exchange matrices with the aid of recombinant enhanced green fluorescent protein overexpressed in Escherichia coli.

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.

Penicillin acylase purification with the aid of hydrophobic charge induction chromatography.

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.

Evaluation of chromatographic recycling for imidazole used in the chromatographic purification of His-tag recombinant proteins.

The aim of this work was to test a recycling method for imidazole used in immobilized metal affinity chromatography (IMAC) as eluent for recombinant histidine-tag (His-tag) protein. After evaluating two supports, the method was optimized with a mixture of bovine serum albumin, sodium chloride and imidazole. Recycling was performed with an eluate fraction from IMAC of His-tag enhanced green fluorescent protein produced in our laboratory and pure imidazole was recovered in water and was analyzed after being freeze-dried. The imidazole was then reused as eluent in IMAC without any modification in its structure or behavior. This procedure can be used for large-scale chromatography.

Cloning, expression and two-step purification of recombinant His-tag enhanced green fluorescent protein over-expressed in Escherichia coli.

In this report, we describe a two-step chromatographic procedure for the purification of His-tag EGFP by immobilized metal affinity expanded bed adsorption (IMAEBA) as the capture step and size exclusion chromatography as the polishing step. The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms. This meets the needs of scientific researchers as well as industrialists in purifying recombinant proteins. The procedure described allowed the obtention of 230 mg pure EGFP from 1 l simple batch culture with a recovery of 90%.

On-column refolding of an insoluble histidine tag recombinant exopolyphosphatase from Trypanosoma brucei overexpressed in Escherichia coli.

An exopolyphosphatase gene has been cloned by polymerase chain reaction (PCR) from Trypanosoma brucei and the corresponding protein overexpressed as a recombinant His-tag (histidine tag) exopolyphosphatase in E. coli in order to characterize its biochemical activity and to produce antibody to determine its localization. Because overexpression of this protein in bacteria resulted in the formation of inactive inclusion bodies, these structures were first solubilized in denaturant condition (6 M urea). Secondly, after a capture step using immobilized metal affinity chromatography (IMAC), a gradual refolding of the protein was performed on-column from 6 M to 0 M urea in the presence of 1% Triton X-100. Triton X-100 was used to abolish protein aggregation during the refolding step. The purified enzyme was active, demonstrating that at least part of the proteins was properly refolded.

Modification of flavonoid using lipase in non-conventional media: effect of the water content.

The enzymatic acylation of a flavonoid (naringin) was investigated in this work. This atypic substrate for a lipase was esterified very selectively by the immobilized Candida antarctica lipase: a single product was synthesized and was assumed to be the 6-O-palmitate naringin ester acylated on the glucose moiety. As lipase-catalyzed esterification reactions in organic media are greatly influenced by the water content, the effect of the initial hydration level of the reaction medium components was pointed out for naringin palmitate synthesis. 2-Methyl 2-butanol (solvent) and naringin (acyl acceptor) provided high amounts of water and when dried increased the conversion yield by 63% and the specific activity by 60%. On the contrary, the enzyme must not be dried because water is essential for the three-dimensional structure of the protein and, if absent, results in a 67% loss of activity. As water was produced in parallel to ester synthesis, the equilibrium of the reaction might be shifted by its removal. When the reaction was carried out with 100 g l(-1) molecular sieves 4A added after 24 h of reaction, a conversion yield of 43% was reached after 55 h reaction.

On-line purification of His-tag enhanced green fluorescent protein taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption.

In this report, we describe a new process for the on-line purification of His-tag EGFP (enhanced green fluorescent protein) taken directly from a bioreactor by continuous ultrasonic homogenization coupled with immobilized metal affinity expanded bed adsorption (IMAEBA). The use of proteins including a histidine-tag facilitates their subsequent purification after expression in many microorganisms. This meets the needs of scientific researchers as well as industrialists interested in purifying recombinant proteins. After evaluating the different flow-rates and ultrasonic probe sizes, the on-line purification was tested. After ultrasonic treatment, 70% of the cells were broken and 90% of free EGFP was recovered after IMAEBA. In our conditions, more than 450 mg of EGFP were obtained in 15 h. On-line bioreactor-ultrasonic probe-immobilized metal affinity expanded bed adsorption is a rapid automated technique for obtaining large quantities of pure EGFP.

Penicillin acylase purification with the aid of pseudo-affinity chromatography.

The introduction of affinity chromatography has opened a new dimension in protein purification. This article reviews the current techniques used in the penicillin acylase purification process, especially pseudo-affinity chromatography. A profile for a suitable ligand is established. An aromatic ring and the presence of one or several amino groups seem essential for proper interaction. Immobilized metal affinity chromatography now seems to be a good competitor.

A novel C-terminal kinesin is essential for maintaining functional acidocalcisomes in Trypanosoma brucei.

Kinesins are cytoskeletal motor proteins that play roles in a variety of fundamental cellular processes including cell division and the anterograde transport of vesicles and organelles. We purified, cloned, and functionally characterized in Trypanosoma brucei a new member of the C-terminal kinesin family, TbKIFC1. Kinetic constants of the recombinant motor domain of TbKIFC1 were estimated at 0.56 microm for the microtubule dissociation constant (K(d)) with a k(cat) of 0.2 s(-1). Immunolocalization analysis showed an association of TbKIFC1 with punctate structures. Because they were rapidly transported to the negative pole of the microtubule after NH(4)Cl treatment, these structures were considered to be associated with acidic vesicles. To determine the role of the kinesin in vivo, we produced an inducible kinesin-deficient strain by double-stranded RNA interference methodology. Mutant cells were loaded with the fluorescent reagent fura2/acetoxymethylester to measure intracellular free calcium ([Ca(2+)](i)). The resting [Ca(2+)](i) was unchanged in mutant cells; however, alkalinization of acidic vesicles induced by NH(4)Cl or nigericin was not followed by release of Ca(2+). These data and the relative importance of the ionomycin-releasable and the ionomycin-plus-NH(4)Cl-releasable Ca(2+) pools suggest a lower Ca(2+) content in acidocalcisomes and dysfunctional Ca(2+) release.

Evaluation of immobilized metal affinity chromatography for purification of penicillin acylase.

The aim of this work was to test immobilized metal affinity chromatography (IMAC) for the purification of penicillin acylase. After evaluation of different metals, Cu2+ was selected. Different samples were tested: pure penicillin acylase, industrial clarified feedstock and crude extract. After comparing two eluents, NH4Cl and imidazole, it appeared that although both gave good results for recovery and activity, NH4Cl was a more selective eluent with a higher fold purification than imidazole (4.64 versus 2.04). Moreover, we shown that a multistep gradient of NH4Cl, greatly increased the degree of purification (12.36) compared with the one-step process as control (4.64). In addition, good recovery was obtained (97-100%).

One-step chromatographic purification procedure of a His-tag recombinant carboxyl half part of the HTLV-I surface envelope glycoprotein overexpressed in Escherichia coli as a secreted form.

A His-tag recombinant carboxyl half part of the HTLV-I surface envelope glycoprotein was overexpressed in E. coli as a secreted form in order to study its biochemical properties and to determine its three-dimensional structure by X-ray crystallography. Starting from several hundred milliliters of culture, a centrifugation was used to eliminate the cells. After solubilization and centrifugation, the protein was then purified by a one-step chromatographic purification procedure. Immobilized Metal Affinity Chromatography (IMAC) was performed by evaluating the tri-dentate iminodiacetic acid (IDA) chelating group with chelating Sepharose fast flow, and the tetra-dendate nitrilotriacetic acid (NTA) chelating group with NTA-agarose. The latter was the most suitable gel for our protein. This expression system and the use of affinity chromatography is a rapid technique to obtain a soluble protein for use in structural studies to further understand the mechanisms of HTLV-1 entry into target cells.