The presence of a common downstream box enables the simultaneous expression of multiple proteins in an E. coli extract

In our experiments to produce different combinations of recombinant proteins in a cell-free protein synthesis system derived from Escherichia coli, we found that certain pairs of ORFs were not expressed equally. Instead, only a single DNA species was expressed dominantly, while the expression of the others was almost completely repressed. This bias during the co-expression of the DNA pairs was eliminated when an identical downstream box sequence was added to the 5'-ends of the template DNA pairs. By introducing identical nucleotide sequences of the his-tag or the downstream box of chloramphenicol acetyltransferase (CAT-DB) in front of the target genes that were otherwise not expressed compatibly, both of the encoded proteins were produced at similar productivities. Moreover, in the presence of a common downstream box, multiple genes were simultaneously expressed in the same reaction mixture. We expect that the proposed approach will offer a powerful tool for the preparation of unbiased protein libraries, as well as for studying the structure and functions of interacting proteins.     

Functional monolithic platforms: Chromatographic tools for antibody purification

Polymer monoliths are an efficient platform for antibody purification. The use of monoclonal antibodies (mAbs) and engineered antibody structures as therapeutics has increased exponentially over the past few decades. Several approaches use polymer monoliths to purify large quantities of antibody with defined clinical and performance requirements. Functional monolithic supports have attracted a great deal of attention as they offer practical advantages for antibody purification, such as more rapid analysis, smaller sample volume requirements and the opportunity for a greater target molecule enrichment. This review focuses on the development of synthetic and natural polymer-based monoliths for antibody purification. The materials and methods employed in monolith production are discussed, highlighting the properties of each system. We also review the structural characterization techniques available using monolithic systems and their performance under different chromatographic approaches to antibody capture and release. Finally, a summary of monolithic platforms developed for antibody separation is presented, as well as expected trends in research to solve current and future challenges in this field. This review comprises a comprehensive analysis of proposed solutions highlighting the remarkable potential of monolithic platforms.     

Multiobjective evolutionary optimization in antibody purification process design

To contribute towards designing more cost-efficient, robust and flexible downstream processes for the manufacture of monoclonal antibodies (mAbs), a framework consisting of an evolutionary multiobjective optimization algorithm (EMOA) linked to a biomanufacturing process economics model is presented. The EMOA is tuned to discover sequences of chromatographic purification steps and column sizing strategies that provide the best trade-off with respect to multiple objectives including cost of goods per gram (COG/g), robustness in COG/g, and impurity removal capabilities. Additional complexities accounted for by the framework include uncertainties and constraints. The framework is validated on industrially relevant case studies varying in upstream and downstream processing train ratios, annual demands, and impurity loads. Results obtained by the framework are presented using a range of visualization tools, and indicate that the performance impact of uncertainty is a function of both the level of uncertainty and the objective being optimized, and that uncertainty can cause otherwise optimal processes to become suboptimal. The optimal purification processes discovered outperform the industrial standard with, e.g. savings in COG/g of up to 10%. Guidelines are provided for choosing an optimal purification process as a function of the objectives being optimized and impurity levels present.     

Ultrasound-assisted production of highly-purified β-glucan schizophyllan and characterization of its immune properties

In this work, we suggest procedure for manufacturing of highly pure schizophyllan (SPG) cleaved into shorter fragments by ultrasonication. The ultrasonicated SPG (uSPG) was prepared from Schizophyllum commune submerged cultivation in bioreactor, culture broth ultrasonication with 100 Ws/mL energy input, followed by depth filtration and ultrafiltration. The uSPG contained less than 0.3%w/w protein and 0.078 IU/mg endotoxin. Chemical composition and structure of the uSPG were analyzed by GC and NMR, and conformation of the molecules was documented by AFM. Apyrogenicity of uSPG was proved by MAT test. Immunomodulatory properties of native and denatured uSPG were studied in vitro with human whole blood. The LPS-spiked samples revealed immunomodulatory activity of both native and denatured uSPG. Protein array showed that native uSPG activates certain cytokines and chemokines significantly stronger than denatured uSPG.     

Protein refolding using chemical refolding additives

In laboratories and manufacturing settings, a rapid and inexpensive method for the preparation of a target protein is crucial for promoting resesrach in protein science and engineering. Inclusion-body-based protein production is a promising method because high yields are achieved in the upstream process, although the refolding of solubilized, unfolded proteins in downstream processes often leads to significantly lower yields. The most challenging problem is that the effective condition for refolding is protein dependent and is therefore difficult to select in a rational manner. Accordingly, considerable time and expense using trial-and-error approaches are often needed to increase the final protein yield. Furthermore, for certain target proteins, finding suitable conditions to achieve an adequate yield cannot be obtained by existing methods. Therefore, to convert such a troublesome refolding process into a routine one, a wide array of methods based on novel technologies and materials have been developed. These methods select refolding conditions where productive refolding dominates over unproductive aggregation in competitive refolding reactions. This review focuses on synthetic refolding additives and describes the concepts underlying the development of reported chemical additives or chemical-additive-b     

Simulation for the recovery of plasmid for a DNA vaccine

The pIDKE2 plasmid is the main component of the CIGB's candidate vaccine against Hepatitis C virus (HVC), which is being used in HCV chronically-infected individuals during clinical trials phase 1 and 2. The designed downstream process of pIDKE2 plasmid produces up to 179 g/year. In order to conduct further improvements, modelling of the downstream process was performed. A methodology based on process analysis tools, such as experimental design and modelling was established to identify factors with the highest influence on production cost and the amount of annual plasmid. Taking into account that the pIDKE2 downstream process designed is in its initial stages of development, CIM technology was evaluated as a new manufacturing process on lab scale. Purity and recovery of CIM technology was better than porous particle matrix, thus SuperPro Designer was used in order to simulate the purification process. Cost efficiency optimization of the pIDKE2 downstream process was done with the simulation model.     

Linear programming as a tool in life cycle assessment

Linear Programming (LP) is a powerful mathematical technique that can be used as a tool in Life Cycle Assessment (LCA). In the Inventory and Impact Assessment phases, in addition to calculating the environmental impacts and burdens, it can be used for solving the problem of allocation in multiple-output systems. In the Improvement Assessment phase, it provides a systematic approach to identifying possibilities for system improvements by optimising the system on different environmental objective functions, defined as burdens or impacts. Ultimately, if the environmental impacts are aggregated to a single environmental impact function in the Valuation phase, LP optimisation can identify the overall environmental optimum of the system. However, the aggregation of impacts is not necessary: the system can be optimised on different environmental burdens or impacts simultaneously by using Multiobjective LP. As a result, a range of environmental optima is found offering a number of alternative options for system improvements and enabling the choice of the Best Practicable Environmental Option (BPEO). If, in addition, economic and social criteria are introduced in the model, LP can be used to identify the best compromise solution in a system with conflicting objectives. This approach is illustrated by a real case study of the borate products system. An erratum to this article is available at .     

Impacts of deep open drains on water quality and biodiversity of receiving waterways in the Wheatbelt of Western Australia

Extensive networks of deep drains are being built in Western Australia to reduce the effects of dryland salinity on agricultural lands. Most of these drains discharge into natural river and wetland systems, with little consideration given to the environmental impacts. This study examined the downstream ecological impacts of one of the oldest deep drain networks in Western Australia, located in the Wakeman subcatchment near Narembeen. Twelve sites were sampled bi-monthly from October 2004 to September 2006. On each occasion, water quality parameters were measured and the macro-invertebrate fauna was sampled. Significant differences in water quality and macro-invertebrates were observed between the untreated sites and those affected by the drain discharge. Surface water at untreated sites was always fresh (<3 ppt), alkaline (pH 7.6–8.9) and turbid (49–600 NTU), whereas treatment sites were always saline (28–147 ppt), acidic (pH 1.9–3.8) and mostly clear (0–100 NTU). No recovery of water quality was observed with distance from discharge point (20 km). Invertebrates reflected differences in water quality, with drain discharge resulting in a sharp decline in species richness, and significant changes in macro-invertebrate community composition. Sites affected by drain discharge were dominated by fly larvae such as Orthocladiinae and Ceratopogonidae. Microcrustaceans were far more abundant at sites unaffected by drainage. The ecological values of Wheatbelt streams are likely to be further compromised by discharge of poor water quality from deep drainage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: K. Martens     

Selection and optimization of an aqueous two‐phase system for the recovery of 1,3‐propandiol from fermentation broth

In this study a suitable alcohol/salt aqueous two‐phase (ATP) system was selected for the recovery of 1,3‐propandiol (1,3‐PD) from fermentation broth. From the different alcohol/salt systems studied the ethanol and dipotassium hydrogen phosphate ATP system appeared to be favorable. To examine the potential of this ATP system the partition coefficient of 1,3‐PD in synthetic solutions was first optimized with the response surface methodology. The parameters studied were concentrations of ethanol (21.99–38.81% w/w), dipotassium hydrogen phosphate (14.99–31.81% w/w) and 1,3‐PD (6.36–73.64 g/L). The optimum conditions were found to be 35.39% w/w for ethanol, 28.40% w/w for dipotassium hydrogen phosphate and 73.6 g/L for 1,3‐PD. Under these conditions the maximum partition coefficient of 1,3‐PD and the extraction yield were determined as 23.14 and 97.82%, respectively. The optimum extraction conditions were then used to guide the recovery of 1,3‐PD from a real fermentation broth. The partition coefficient and extraction yield of 1,3‐PD reached 20.28–97.20% in this case, respectively. A favorable partition of the organic acids lactate, acetate and butyrate in the bottom phase was also achieved. We have also studied the removal of cells and macromolecules from the broth. Removal ratio of cells and proteins were 96.47 and 93.05%, respectively. Thus, the ethanol/dipotassium hydrogen phosphate ATP system appears to be an interesting alternative or can be used as one useful step in the downstream processing of 1,3‐PD from fermentation broth.