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While the miniaturization of the High Throughput Screening (HTS) process has allowed the scaling up of the number of wells/plate from 96 to 384 and up to 1536 and a saving in the amount of compound libraries used for screening, the low number of cells/well means the reporter cells are forced to generate a strong and reproducible signal that can be accurately detected.
Axxam is developing several tools to compensate for the difference between the biopharmaceutical industry's need for low-volume screening and the signal weakness of currently-available tools.
Our researchers focus on the development of new reporter genes and cell lines to overcome these problems, such as:
New Green Fluorescence Proteins
The first Green Fluorescence Protein (GFP) was isolated by the bioluminescent jellyfish Aequorea Victoria that emits green fluorescence when irradiated with long-wave ultraviolet light.
The heterologous expression of GFP had proven that its maturation and formation of chromophore was independent of factors such as enzymes, other proteins, external substrates or co-factors with exception of molecular oxygen. In fact, the gene encodes a chromophore intrinsically within its protein sequence, excluding and enabling a strong fluorescence activity.
These features paved the way for GFP to become an important tool in molecular and cellular biology as a transcriptional reporter, fusion tag, partner for fluorescence resonance energy transfer (FRET) and biosensor.
Despite its remarkable usefulness, wildtype GFP exhibits some limitations due to problems with expression level, photosensitivity, low brightness, insolubility and the tendency to dimerize.
To overcome these reported disadvantages, the Axxam Enabling Technologies group concentrates on the molecular characterization of
new GFPs, isolated and cloned at Axxam, from other marine organisms. They are being carefully characterized with particular attention to the oligomerization problem. Oligomerization does not narrow the usage of these proteins as reporter genes or biomarkers but does preclude their use in fusion protein applications since they tend to form bulky products which could lead to interferences of translocation of the target protein.
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