Highly dense beaded sorbents suitable for fluidized bed applications

New highly dense beaded sorbents suitable for fluidized bed applications of protein separations are presented. They are prepared using porous mineral oxides supporting functional hydrogels responsible for protein interaction. Beads of small diameter (70m) are selected to reduce mass transfer resistance. Zirconium oxide was the preferred mineral material due to its high density (5.9g/ml) allowing high fluidizing liquid velocities (600 cm/h) into columns with a moderate bed expansion (lower than 3). Composite mineral - hydrogel sorbents are evaluated for their ability to rapidly adsorb proteins in fluidized bed and to separate with an appropriate resolution macromolecule mixtures in packed bed. Lysozyme dynamic capacities of 68 and 53 mg per ml of sedimented bed were obtained at fluidizing velocities of 450 and 900 cm/h.     

Surface-enhanced laser desorption-ionization retentate chromatography mass spectrometry (SELDI-RC-MS): a new method for rapid development of process chromatography conditions

Protein biochip arrays carrying functional groups typical of those employed for chromatographic sorbents have been developed. When components of a protein mixture are deposited upon an array's functionalized surface, an interaction occurs between the array's surface and solubilized proteins, resulting in adsorption of certain species. The application of gradient wash conditions to the surface of these arrays produces a step-wise elution of retained compounds akin to that accomplished while utilizing columns for liquid chromatography (LC) separations. In retentate chromatography-mass spectrometry (RC-MS), the "retentate" components that remain following a wash are desorbed and ionized when a nitrogen laser is fired at discrete spots on the array after treatment with a laser energy-absorbing matrix solution. Ionized components are analyzed using a time-of-flight mass spectrometer (TOF MS). The present study demonstrates that protein biochips can be used to identify conditions of pH and ionic strength that support selective retention-elution of target proteins and impurity components from ion-exchange surfaces. Such conditions give corresponding behavior when using process-compatible chromatographic sorbents under elution chromatography conditions. The RC-MS principle was applied to the separation of an Fab antibody fragment expressed in Escherichia coli as well as to the separation of recombinant endostatin as expressed in supernatant of Pichia pastoris cultures. Determined optimal array binding and elution conditions in terms of ionic strength and pH were directly applied to regular chromatographic columns in step-wise elution mode. Analysis of collected LC fractions showed favorable correlation to results predicted by the RC-MS method.     

The membrane form of acetylcholinesterase from rat brain contains a 20 kDa hydrophobic anchor

Rat brain acetylcholinesterase (AChE, EC 3.1.1.7) consists of about 80% amphiphilic detergent-soluble (DS-) AChE and 20% hydrophilic salt-soluble (SS-) AChE. DS-AChE contains about 65% tetrameric, 20% dimeric and 10% monomeric, SS-AChE about 40% tetrameric and 60% monomeric forms. N-terminal sequencing of DS- and SS-AChE gave identical N-termini corresponding to the published cDNA sequence of the mature enzyme. The band pattern on SDS-gels is similar to that of AChE from human and bovine brain. SDS-PAGE of hydrophobically labeled DS-AChE revealed the presence of a disulfide bonded hydrophobic membrane anchor of about 20 kDa. Monoclonal antibodies (mAbs) recognizing the anchor-containing subunits of mammalian brain DS-AChE, crossreacted with rat brain DS-AChE but not with SS-AChE. DS- and SS-AChE also reacted with antibodies raised against a peptide comprising the last 10 amino acids of the sequence of bovine brain AChE. Our results led us to conclude that both DS- and SS-AChE from rat brain contain T-type catalytic subunits, and DS-AChE in addition a P-type hydrophobic anchor similar to other mammalian brain DS-AChE.     

Intrinsic membrane proteins of the thylakoids of Chlamydomonas reinhardii

Upon protoolytle treatment of thylakoid membranes, extrinsic proteins are selectively degraded. The remaining resistant proteins have been analyzed by SDS-PAGE. In the thylakoids of Chlamydomonas reinhardii, six polypeptides or protein fragments of 20 kD or higher are resistant to proteolysis. These intrinsic proteins have been identified as: the apoproteins of the chlorophyll-protein complexes CP I and LHCP; a polypeptide whose presence is related to the chlorophyll b content of the cells; and a portion of a chloroplast-made 34 kD polypeptide. Furthermore, after proteolytic treatment of the membranes, the LHCP complex can be resolved into two subcomplexes, apparently differing in their polypeptide composition.

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Zusammenfassung Durch proteolytische Behandlung von Thylakoidmembranen worden die extrinsischen Proteinanteile selektiv abgebaut. Die resistenten, in der Membran verbleibenden Polypeptide können mit SDS-PAGE analysiert werden. In Thylakoiden von Chlamydomonas reinhardii finden sich sechs resistente Polypeptide mit Molekulargewichten über 20'000. Durch quantitative Bestimmung während der Proteolyse und durch limitierte Fragmentierung nach Cleveland wurden die resistenten Polypeptide identifiziert als: (a) Apoprotein von CP 1, (b) Apoproteine des LHCP-Komplexes, (c) Teil des chloroplastidial synthetisierten 34 kD-Proteins und (d) Polypeptid, welches möglicherweise mit dem Chlorophyll b-Gehalt in Verbindung steht. Im weiteren führte die proteolytische Behandlung der Thylakoide zu einer Auftrennung des LHCP-Komplexes in zwei Subkomplexe mit unterschiedlicher Proteinzusammensetzung.

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Abbreviations CP 1 pigment-protein complex with slower mobility in SDS electrophoresis, corresponding to the P700 chlorophyll a protein complex - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - EDTA ethylene-diaminotetraacetic acid - kD kilo Dalton - LHCP pigment-protein complex with intermediate mobility in SDS electrophoresis, corresponding to the light-harvesting chlorophyll a/b protein complex - SDS sodiumdodecyl sulfate - SDS-PAGE electrophoresis in SDS-containing polyacrylamide gels - Tris tris (hydroxymethyl) aminomethane     

Chloroplasts of the green alga Chlamydomonas reinhardtii possess at least four distinct stromal processing proteases

The majority of the proteins in the chloroplast are encoded in the nucleus and synthesised in the cytoplasm as precursors with N-terminal extensions. These targeting sequences guide the precursor proteins into the chloroplast where they are immediately cleaved off by a stromal processing protease (SPP). It is commonly assumed that in higher plant chloroplasts one general SPP processes almost all imported precursor proteins. In the green alga Chlamydomonas, however, there exist several different SPPs which process the various Chlamydomonas precursor proteins. The seven precursor proteins investigated here, which were all correctly imported into isolated chloroplasts, could be divided into two groups: Four precursor proteins were cleaved correctly when processed in vitro with an extract of stromal proteins. Four different SPPs were found in Chlamydomonas chloroplasts to be responsible for the processing of this class of precursors and these four activities were separated chromatographically, characterised and further distinguished by their sensitivity to different inhibitors. The three precursors of the second group were degraded completely by unidentified enzyme(s) present in the stromal extract. Degradation of these precursors was dependent on their conformational integrity as well as on the redox state in the stroma. This revised version was published online in June 2006 with corrections to the Cover Date.     

2-Mercapto-5-benzimidazolesulfonic acid: an effective multimodal ligand for the separation of antibodies

The report describes the use of 2-mercapto-5-benzimidazolesulfonic acid (MBISA) as a ligand for the separation of antibodies by chromatography. The ligand shows a relatively specific adsorption property for antibodies from very crude biologicals at pH 5.0-5.5. At this pH range most of other proteins do not interact with the resin especially when the ionic strength is similar to physiological conditions. Several characterization studies are described such as antibody adsorption in different conditions of ionic strength, pH and temperature. These properties are advantageously used to selectively capture antibodies from very crude feed stocks without dilution or addition of lyotropic salts. Demonstration was made that the adsorption mechanism is neither based on ion exchange nor on hydrophobic associations, but rather as an assembly of a variety of properties of the ligand itself. Binding capacity in the described conditions ranges between 25 and 30 mg/mL of resin. The sorbent does not co-adsorb albumin (Alb) and seems compatible with a large variety of feedstocks. Quantitative antibody desorption occurs when the pH is raised above 8.5. The final purity of the antibody depends on the nature of the feedstock, and can reach levels of purity as high as 98%. Even with very crude biological liquids such as ascites fluids, cell culture supernatants and Chon fraction II + III from human plasma fractionation where the number of protein impurities is particularly large, immunoglobumins G (IgG) were separated at high purity level in a single step.