A comparison of Lyse-It to other cellular sample preparation, bacterial lysing, and DNA fragmentation technologies.

The ability for safe and rapid pathogenic sample transportation and subsequent detection is an increasing challenge throughout the world. Herein, we describe and use bead-beating, vortex, sonication, 903 protein saver cards, and Lyse-It methods, aiming to inactivate Gram-positive and -negative bacteria with subsequent genome DNA (quantitative Polymerase Chain Reaction) qPCR detection. The basic concepts behind the four chosen technologies is their versatility, cost, and ease of use in developed and underdeveloped countries. The four methods target the testing of bacterial resilience, cellular extraction from general and complex media and subsequent DNA extraction for qPCR detection and amplification. These results demonstrate that conventional high temperature heating, 903 protein saver cards, and Lyse-It are all viable options for inactivating bacterial growth for safe shipping. Additionally, Lyse-It was found to be particularly useful as this technology can inactivate bacteria, extract cells from 903 protein saver cards, lyse bacterial cells, and additionally keep genomic DNA viable for qPCR detection.

Integrated process development-a robust, rapid method for inclusion body harvesting and processing at the microscale level.

Escherichia coli stores large amounts of highly pure product within inclusion bodies (IBs). To take advantage of this beneficial feature, after cell disintegration, the first step to optimal product recovery is efficient IB preparation. This step is also important in evaluating upstream optimization and process development, due to the potential impact of bioprocessing conditions on product quality and on the nanoscale properties of IBs. Proper IB preparation is often neglected, due to laboratory-scale methods requiring large amounts of materials and labor. Miniaturization and parallelization can accelerate analyses of individual processing steps and provide a deeper understanding of up- and downstream processing interdependencies. Consequently, reproducible, predictive microscale methods are in demand. In the present study, we complemented a recently established high-throughput cell disruption method with a microscale method for preparing purified IBs. This preparation provided results comparable to laboratory-scale IB processing, regarding impurity depletion, and product loss. Furthermore, with this method, we performed a "design of experiments" study to demonstrate the influence of fermentation conditions on the performance of subsequent downstream steps and product quality. We showed that this approach provided a 300-fold reduction in material consumption for each fermentation condition and a 24-fold reduction in processing time for 24 samples.

Effect of ion and surfactant choice on the recovery of a histidine-tagged protein from tobacco extract using foam fractionation.

Tobacco plants can be used for the production of proteins for pharmaceutical applications. One of the most difficult and expensive tasks associated with this technology is isolating the product of interest from the hundreds of other chemicals found in tobacco. We describe a new recovery strategy in which the protein of interest is "tagged" with a histidine structure, which forms a complex with metal ions and a surfactant that will accumulate in the foamate of a foam fractionation step. His-gus, a histidine-tagged enzyme, was selectively recovered in the presence of two different surfactants and two different metal ions. The foam fractionation with N-epsilon-dodecylamido-N-alpha, N-alpha,-bis(carboxymethyl)-L-lysine surfactant and Ni2+ ions resulted in an average His-gus activity recovery value of 88% and an activity enrichment of 2.27. The performance of the recovery strategy without tobacco extract resulted in an average activity recovery value of 63.32% and an average activity enrichment value of 5.16, utilizing lauroyl ethylenediaminetriacetate surfactant and Ni2+ ions. It was shown that even though a majority of the native tobacco proteins are removed during the prefoaming step, the presence of tobacco extract does affect the recovery of His-gus.