Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5''-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 °C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.     

Impact of clarification strategy on chromatographic separations: Pre-processing of cell homogenates

This research focused on how the extent and type of primary solid-liquid separation can affect the performance of guard filtration and chromatography, in this instance hydrophobic interaction chromatography. The system used in the study was yeast (Saccharomyces cerevisiae) with the target molecule being an intracellular protein; alcohol dehydrogenase (ADH). As expected, loading more poorly clarified suspensions (both centrates and primary filtrates) required proportionally larger guard filtration areas. In addition for feed suspensions prepared by centrifugation, increased clarification led to greater column capacity. However, where filtration was used to achieve similar clarification considerably lower column capacity was achieved. These results were attributed to centrifugation leading to the aggregation of lipids and their subsequent removal in this form before application to the column. Clarification by filtration leaves such lipids in their original "soluble" state and hence they are not removed. The importance of the need to examine such interactive effects in bioprocess studies is discussed. This observation was confirmed with further analytical work into the nature of the aggregated material formed in the supernatant under centrifugation conditions. This material was only soluble in an organic solvent, and identified as phophatidylcholine and ergosterol as among the components removed by centrifugation and guard filtration as opposed to filtration and guard filtration.     

Effects of solution environment on mammalian cell fermentation broth properties: Enhanced impurity removal and clarification performance

The processing of recombinant proteins from high cell density, high product titer cell cultures containing mammalian cells is commonly performed using tangential flow microfiltration (MF). However, the increased cellular debris present in these complex feed streams can prematurely foul the membrane, adversely impacting MF capacity and throughput. In addition, high cell density cell culture streams introduce elevated levels of process‐related impurities, which increase the burden on subsequent purification operations to remove these complex media components and impurities. To address this challenge, an evaluation of mammalian cell culture broth buffer properties was examined to determine if enhanced impurity removal and clarification performance could be achieved. A framework is presented here for establishing optimized mammalian cell culture buffer conditions, involving trade‐offs between product recovery and purification and improved clarification at manufacturing‐scale production. A reduction in cell culture broth pH to 4.7–5.0 induced flocculation and impurity precipitation which increased the average feed particle‐size. These conditions led to enhanced impurity removal and improved MF throughput and filter capacity for several mammalian systems. Feed conditions were further optimized by controlling ionic composition along with pH to improve product recovery from high cell density/high product titer cell cultures. Biotechnol. Bioeng. 2011; 108:50–58. © 2010 Wiley Periodicals, Inc.     

Pectolytic enzymes co-immobilization on γ-alumina spheres via organophosphate compounds

Endopectinlyase (EC 4.2.2.10), endopolygalacturonase (EC 3.2.1.15) and pectinesterase (EC 3.1.1.11) present in a commercial mixture were co-immobilized on γ-alumina spheres activated with organophosphate compounds. Staining of the alumina-enzyme complexes with a dye specific for protein showed that only the carrier external surface was available for protein binding. When confined in a packed bed reactor, the activity of the co-immobilized enzymes brought about a viscosity decrease of 70–90% in pectin and polygalacturonic acid solutions, respectively. Sixty per cent of the initial activity was retained from the immobilized enzymes after the sixth utilization cycle on both the substrates used. The immobilized enzymes were also active against fresh apple juice producing a 90% reduction in viscosity in the first five cycles of utilization.     

Pectin Methylesterase of Datura species,purification, and characterization from Datura stramoniumand its application

Pectin methylesterases (PME; EC 3.1.1.11) involved in de-esterification of pectin and have applicability in food, textiles, wines, pulp, and paper industries. In the present study, we compared PME activity of different parts of 3 Datura species and found that fruit coat showed maximum PME activity followed by leaf and seed. PME from leaves of D. stramonium (DsPME) was purified and characterized. DsPME showed optimum activity at 60 °C and pH 9 in the presence of 0.3 M NaCl. DsPME was stable at 70 °C and retained more than 40% activity after 60 min of incubation. However, enzyme activity completely abolished at 80 after 5 min of incubation. It follows Michaelis-Menten enzyme kinetics. Km and Vmax with citrus pectin were 0.008 mg/ml and 16.96 µmol/min, respectively. DsPME in combination with polygalactourenase (PGA) increased the clarity of orange, apple, pomegranate and pineapple juices by 2.9, 2.6, 2.3, and 3.6 fold, respectively in comparison to PGA alone. Due to very high de-esterification activity, easy denaturation and significant efficacy in incrementing clarification of fruit juice makes DsPME useful for industrial application.     

CLARIFYING APPEALS TO DIGNITY IN MEDICAL ETHICS FROM AN HISTORICAL PERSPECTIVE

Over the past few decades the concept of (human) dignity has deeply pervaded medical ethics. Appeals to dignity, however, are often unclear. As a result some prefer to eliminate the concept from medical ethics, whereas others try to render it useful in this context. We think that appeals to dignity in medical ethics can be clarified by considering the concept from an historical perspective. Firstly, on the basis of historical texts we propose a framework for defining the concept in medical debates. The framework shows that dignity can occur in a relational, an unconditional, a subjective and a Kantian form. Interestingly, all forms relate to one concept since they have four features in common: dignity refers, in a restricted sense, to the 'special status of human beings'; it is based on essential human characteristics; the subject of dignity should live up to it; and it is a vulnerable concept, it can be lost or violated. We argue that being explicit about the meaning of dignity will prevent dignity from becoming a conversation-stopper in moral debate. Secondly, an historical perspective on dignity shows that it is not yet time to dispose of dignity in medical ethics. At least Kantian and relational dignity can be made useful in medical ethics.     

Robust depth filter sizing for centrate clarification

Cellulosic depth filters embedded with diatomaceous earth are widely used to remove colloidal cell debris from centrate as a secondary clarification step during the harvest of mammalian cell culture fluid. The high cost associated with process failure in a GMP (Good Manufacturing Practice) environment highlights the need for a robust process scale depth filter sizing that allows for (1) stochastic batch‐to‐batch variations from filter media, bioreactor feed and operation, and (2) systematic scaling differences in average performance between filter sizes and formats. Matched‐lot depth filter media tested at the same conditions with consecutive batches of the same molecule were used to assess the sources and magnitudes of process variability. Depth filter sizing safety factors of 1.2–1.6 allow a filtration process to compensate for random batch‐to‐batch process variations. Matched‐lot depth filter media in four different devices tested simultaneously at the same conditions was used with a common feed to assess scaling effects. All filter devices showed <11% capacity difference and the Pod format devices showed no statistically different capacity differences. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1542–1550, 2015