Browsing by Subject "E. Coli"
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(2015)Susceptibility to antibiotics is constantly developing in bacteria due to selection pressure caused by use of antibiotics. For this reason, finding new antimicrobial substances is imperative. High-throughput screening (HTS) is an important tool to find new active substances. The need to analyse as many substances in as small time as possible is emphasised in modern drug development. Robust methods, suitable for fast throughput of substances, miniaturisation and automation, are particularly useful. In the context of antimicrobial screening, methods utilising bioluminescence can correspond this need, and genetic engineering can help in developing bacterial strains with beneficial features for screening. In this work, two screening methods were developed and optimised using genetically engineered Escherichia coli strains. The screening methods make use of the bioluminescent properties of the strains, and the methods can be used to screen compound libraries for antimicrobials rapidly enough to approach HTS. The strain E. coli WZM120/pCGLS 11 is constitutively luminescent, so weakening of luminescence means the cell viability weakens. The strain E. coli K12/pCSS305, where luminescence is produced by a heat-inducible runaway plasmid, can be used to especially detect compounds inhibiting DNA replication. In developing the method, workflow was optimised and conditions were validated so as to enable possible HTS campaigns. The target was to create as simple, fast and reproducible a method as possible. The Z' values calculated in assessing the performance are excellent for a cell-based method. The signal is readily distinguishable, the bacterial strains are in a stable manner, and the method is well reproducible. It is possible to continue assay development from 96-well format to 384-well format.
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(2014)Proteins are endogenous molecules that carry out most biological functions in vivo. They are called as the biological workhorses. Proteins are made up of polypeptide chains that usually fold in the three dimensional space to adopt a native stable conformation. Stability of proteins is dependent on the interplay of environmental factors (pH, temperature, ionic strength). For most proteins, the biological function closely relates to the structural attributes of the protein. Misfolding or unfolding of proteins often result in aggregation. Protein aggregation in vivo is known to cause debilitating and fatal diseases such as Alzheimer's, Huntington's, Parkinson's and age related macular degeneration (AMD). Instability (physical and chemical) of proteins in vitro is believed to result in aggregation. This is a huge concern for the biopharmaceutical industry as it not only limits the effectiveness of the manufacturing process but also poses a great risk of fatality in vivo due to the immunogenic nature of the aggregates. Mechanisms of protein aggregation are complex and not well understood. Regulatory requirements for patient safety in biopharmaceutical products require characterization and analysis of aggregates in protein drug formulations. This review provides an overview of protein aggregation in general and highlights the different analytical methods used to characterize protein aggregates in biopharmaceuticals. Neurotrophic factors influence survival, differentiation, proliferation and death of neuronal cells within the central nervous system. Human ciliary neurotrophic factor (hCNTF) has neuroprotective properties and is also known to influence energy balance. Consequently, hCNTF has potential therapeutic applications in neurodegenerative, obesity and diabetes related disorders. Clinical and biological applications of CNTF necessitate a recombinant expression system to produce large amounts of functional protein. Previous studies have reported that recombinant expression of CNTF in Escherichia coli (E. coli) was limited by low yields and the need to refold the protein from inclusion bodies. In this report, we describe a strategy to effectively screen fusion constructs and expression conditions for soluble hCNTF production in E. coli. Most conditions tested with the codon optimized hCNTF sequence in fusion with soluble tags resulted in soluble expression of the protein. The construct 6-His-CNTF showed soluble expression in all the conditions tested. Our results suggest that codon optimization of the hCNTF sequence is sufficient for soluble expression in E. coli. The recombinant hCNTF was found to bind to CNTFRα with an EC50 = 36 nM.
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